Modified L-Asparaginase

ABSTRACT

The disclosure provides a modified protein that is a combination of (i) an L-asparaginase and (ii) one or more (poly)peptide(s), wherein the (poly)peptide consists solely of proline and alanine amino acid residues, and methods of preparation and use thereof.

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename: sequencelisting; daterecorded: Jun. 28, 2017; file size: 34 KB).

BACKGROUND

Proteins with L-asparagine aminohydrolase activity, commonly known asL-asparaginases, have successfully been used for the treatment of AcuteLymphoblastic Leukemia (ALL) in children for many years. ALL is the mostcommon childhood malignant cancers (Avramis (2005) Clin. Pharmacokinet.44, 367-393).

L-asparaginase has also been used to treat Hodgkin's disease, acutemyelocytic Leukemia, acute myelomonocytic Leukemia, chronic lymphocyticLeukemia, lymphosarcoma, reticulosarcoma, and melanosarcoma (Kotzia(2007) J. Biotechnol. 127, 657-669). The anti-tumor activity ofL-asparaginase is believed to be due to the inability or reduced abilityof certain malignant cells to synthesize L-asparagine (Id). Thesemalignant cells rely on an extracellular supply of L-asparagine.However, the L-asparaginase enzyme catalyzes the hydrolysis ofL-asparagine to aspartic acid and ammonia, thereby depleting circulatingpools of L-asparagine and killing tumor cells that cannot performprotein synthesis without L-asparagine (Id).

L-asparaginase from E. coli was the first enzyme drug used in ALLtherapy and has been marketed as Elspar® in the United States or asKIDROLASE and L-asparaginase MEDAC in Europe. L-asparaginases have alsobeen isolated from other microorganisms, e.g., an L-asparaginase proteinfrom Erwinia chrysanthemi, named crisantaspase, that has been marketedas ERWINASE (Wriston (1985) Meth. Enzymol. 113, 608-618; Goward (1992)Bioseparation 2, 335-341). L-asparaginases from other species of Erwiniahave also been identified, including, for example, Erwinia chrysanthemi3937 (Genbank Accession No. AAS67028), Erwinia chrysanthemi NCPPB 1125(Genbank Accession No. CAA31239), Erwinia carotovora (Genbank AccessionNo. AAP92666), and Erwinia carotovora subsp. artroseptica (GenbankAccession No. AAS67027). These Erwinia chrysanthemi L-asparaginases haveabout 91-98% amino acid sequence identity with each other, while theErwinia carotovora L-asparaginases have approximately 75-77% amino acidsequence identity with the Erwinia chrysanthemi L-asparaginases (Kotzia(2007) J. Biotechnol. 127, 657-669).

L-asparaginases of bacterial origin have a high immunogenic andantigenic potential and frequently provoke adverse reactions rangingfrom mild allergic reaction to anaphylactic shock in sensitized patients(Wang (2003) Leukemia 17, 1583-1588). E. coli L-asparaginase isparticularly immunogenic, with reports of the presence ofanti-asparaginase antibodies to E. coli L-asparaginase followingintravenous or intramuscular administration reaching as high as 78% inadults and 70% in children (Id).

L-asparaginases from Escherichia Coli and Erwinia chrysanthemi differ intheir pharmacokinetic properties and have distinct immunogenic profiles,respectively (Klug Albertsen (2001) Brit. J. Haematol. 115, 983-990).Furthermore, it has been shown that antibodies that developed after atreatment with L-asparaginase from E. coli do not cross react withL-Asparaginase from Erwinia (Wang (2003) Leukemia 17, 1583-1588). Thus,L-asparaginase from Erwinia (crisantaspase) has been used as a secondline treatment of ALL in patients that react to E. coli L-asparaginase(Duval (2002) Blood 15, 2734-2739; Avramis (2005) Clin. Pharmacokinet.44, 367-393).

In another attempt to reduce immunogenicity associated withadministration of microbial L-asparaginases, an E. coli L-asparaginasehas been developed that is modified with methoxy-polyethyleneglycol(mPEG) This so-called mPEG-L-asparaginase, or pegaspargase, marketed asONCASPAR (Enzon Inc.), was first approved in the U.S. for second linetreatment of ALL in 1994, and has been approved for first-line therapyof ALL in children and adults since 2006.

ONCASPAR is an E. coli L-asparaginase that has been modified at multiplelysine residues using 5 kDa mPEG-succinimidyl succinate (SS-PEG) (U.S.Pat. No. 4,179,337). SS-PEG is a PEG reagent of the first generationthat contains an unstable ester linkage that is sensitive to hydrolysisby enzymes or at slightly alkaline pH values (U.S. Pat. No. 4,670,417).These properties decrease both in vitro and in vivo stability and canimpair drug safety.

Furthermore, it has been demonstrated that antibodies developed againstL-asparaginase from E. coli will cross react with ONCASPAR (Wang (2003)Leukemia 17, 1583-1588). Even though these antibodies were notneutralizing, this finding clearly demonstrated the high potential forcross-hypersensitivity or cross-inactivation in vivo. Indeed, in onereport 30-41% of children who received pegaspargase had an allergicreaction (Id).

In addition to outward allergic reactions, the problem of “silenthypersensitivity” was recently reported, whereby patients developanti-asparaginase antibodies without showing any clinical evidence of ahypersensitivity reaction (Wang (2003) Leukemia 17, 1583-1588). Thisreaction can result in the formation of neutralizing antibodies to E.coli L-asparaginase and pegaspargase; however, these patients are notswitched to Erwinia L-asparaginase because there are not outward signsof hypersensitivity, and therefore they receive a shorter duration ofeffective treatment (Holcenberg (2004) J. Pediatr. Hematol. Oncol. 26,273-274).

Erwinia chrysanthemi L-asparaginase treatment is often used in the eventof hypersensitivity to E. coli-derived L-asparaginases. However, it hasbeen observed that as many as 30-50% of patients receiving ErwiniaL-asparaginase are antibody-positive (Avramis (2005), Clin.Pharmacokinet. 44, 367-393). Moreover, because Erwinia chrysanthemiL-asparaginase has a shorter elimination half-life than the E. coliL-asparaginases, it must be administered more frequently (Id). In astudy by Avramis et. al, Erwinia asparaginase was associated withinferior pharmacokinetic profiles (Avramis (2007), J. Pediatr. Hematol.Oncol. 29, 239-247). E. coli L-asparaginase and pegaspargase thereforehave been the preferred first-line therapies for ALL over ErwiniaL-asparaginase.

Numerous biopharmaceuticals have successfully been PEGylated andmarketed for many years. However, in many cases, PEGylatedbiopharmaceuticals show significantly reduced activity compared to theunmodified biopharmaceutical. In the case of L-asparaginase from Erwiniacarotovora, it has been observed that PEGylation reduced its in vitroactivity to approximately 57% (Kuchumova (2007) Biochemistry (Moscow)Supplement Series B: Biomedical Chemistry, 1, 230-232). TheL-asparaginase from Erwinia carotovora has only about 75% homology tothe Erwinia chrysanthemi L-asparaginasc (crisantaspace). For ONCASPAR itis also known that its in vitro activity is approximately 50% comparedto the unmodified E. coli L-asparaginase.

Thus, the technical problem underlying the present invention is theprovision of means and methods for treating cancer, such as leukemia ornon-Hodgkin's lymphoma, that avoids the limitations and disadvantages ofprior art therapies, particularly of some PEGylated asparaginases.

The technical problem is solved by provision of the embodimentscharacterized in the claims.

SUMMARY OF THE INVENTION

The present invention relates to a modified protein that is acombination of (i) an L-asparaginase and (ii) one or more(poly)peptide(s), wherein the (poly)peptide consists solely of prolineand alanine amino acid residues. The modified protein can be formed in anumber of ways, including chemical conjugation between theL-asparaginase and the (poly)peptides or by expressing the modifiedprotein as a fusion protein. Also provided herein are nucleic acidsencoding the modified protein, vectors and/or host cells comprisingsame, as well as processes for their production. Compositions comprisingthe modified protein and their use in medicine, particularly in thetreatment of cancer, are disclosed. In another aspect of the invention,the L-asparaginase can be derived from Erwinia and/or it has at least85% identity to the amino acid sequence of SEQ ID NO: 1.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings.

FIG. 1: Chemistry of the conjugation of Crisantaspase with N-terminallyprotected P/A peptides via amino groups.

(A) and (B) depict the chemical structures of P/A peptides (SEQ ID NO:16 and 17, amino acid sequence shown in SEQ ID NO: 5 and 15) containingeither 20 or 40 Pro/Ala residues (respectively), which were obtained bysolid-phase peptide synthesis. In order to avoid polymerization of thepeptides upon chemical activation of the C-terminus, the N-terminus wasprotected with pyroglutamyl (Pga) residue Aminohexanoic acid (Ahx) wasincorporated at the C-terminus of the peptides to serve as linker. (C)In the presence of the non-nucleophilic base N,N-diisopropylethylamine(DIPEA, Hünig's base), and with DMSO as solvent, the N-terminallyprotected P/A peptide is activated at its C-terminus with thebenzotriazol derivativeO-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU). The hydroxybenzotriazol (HOBt) active ester of the peptide issubsequently used to derivatize the amino groups (ε-amino groups oflysine residues or α-amino group of N-terminus) of Crisantaspase withthe P/A peptide through formation of peptide or isopeptide bonds, whilefree HOBt is released. This coupling step is performed in aqueoussolution (e.g. PBS buffer) with a content of organic solvent ≤30%. TheP/A-Crisantaspase modified protein may be purified from residual P/Apeptide/coupling reagent by dialysis and/or chromatography (e.g. ionexchange chromatography).

FIG. 2: Optimization of Crisantaspase/Pga-P/A(20)-Ahx coupling ratio.

Recombinant Crisantaspase produced in E. coli was conjugated with thePga-P/A#1(20)-Ahx peptide (Part A of FIG. 1)(SEQ ID NO: 16, amino acidsequence shown in SEQ ID NO: 5) as described in Example 1. Thepeptide-to-protein ratio was varied between 3.5 mg and 10 mg P/A peptideper 1 mg Crisantaspase. The gel was loaded with 7 μg of Crisantaspasefrom each coupling reaction. Additionally, a mix of coupling reactionswith ratios of 0.3 to 10 mg peptide per mg protein was applied as sizestandard (“Std”). The number of coupled P/A peptides can be determinedby counting the bands in that ladder starting from the unconjugatedCrisantaspase as marked on the right. Lane “kDa”: PIERCE UnstainedProtein MW Marker (Thermo Fisher Scientific).

FIG. 3: Purification of Crisantaspase/Pga-P/A(40)-Ahx peptide couplingproduct via anion exchange chromatography

Recombinant Crisantaspase produced in E. coli was conjugated with thePga-P/A(40)-Ahx peptide (Part B of FIG. 1) (SEQ ID NO: 17, amino acidsequence shown in SEQ ID NO: 15) as described in Example 2. Afterdialysis against AIX running buffer (25 mM boric acid/NaOH pH 9.0, 1 mMEDTA) anion exchange chromatography was performed on an 85 mL SOURCE 15Qcolumn (A). By applying an NaCl concentration gradient, the enzymemodified protein eluted in a single sharp peak, as revealed by the UVtrace at 280 nm. Separation of remaining uncoupled peptide and othernon-proteinous byproducts of the chemical conjugation devoid of UVabsorption at 280 nm was monitored by the 225 nm UV trace. (B) SDS-PAGEanalysis of the Crisantaspase/Pga-P/A(40)-Ahx modified protein afterpurification by anion exchange chromatography (lane 1). A mix ofcoupling reactions with ratios of 0.3 to 10 mg peptide per mg proteinwas applied to lane 2 to allow determination of the number of coupledP/A peptides per Crisantaspase monomer. PAGERULER Plus Prestained marker(Thermo Fisher Scientific) was applied to lane “M”.

FIG. 4: Purification of Crisantaspase/Pga-P/A(20)-Ahx peptide couplingproduct via anion exchange chromatography

Recombinant Crisantaspase produced in E. coli was conjugated with thePga-P/A(20)-Ahx peptide (Part A of FIG. 1) (SEQ ID NO: 16, amino acidsequence shown in SEQ ID NO: 5) as described in Example 3. Afterdialysis against AIX running buffer (25 mM boric acid/NaOH pH 9.0, 1 mMEDTA) anion exchange chromatography was performed on an 85 mL SOURCE 15Qcolumn (A). By applying an NaCl concentration gradient the enzymemodified protein eluted in a single sharp peak, as revealed by the UVtrace at 280 nm. Separation of remaining uncoupled peptide and othernon-proteinous byproducts of the chemical conjugation devoid of UVabsorption at 280 nm was revealed by the 225 nm UV trace. (B) SDS-PAGEanalysis of the Crisantaspase/Pga-P/A(20)-Ahx modified protein afterpurification by anion exchange chromatography (lane 1). A mix ofcoupling reactions with ratios of 0.3 to 10 mg peptide per mg proteinwas applied to lane 2 to allow determination of the number of coupledP/A peptides per Crisantaspase monomer. PAGERULER Plus Prestained marker(Thermo Fisher Scientific) was applied to lane “M”.

FIG. 5: Cloning of the expression vectors for the production ofPASylated Crisantaspase in E. coli

(A) Plasmid map of pASK75-SapI-Crisantaspase (SEQ ID NO: 4) and (B) ofits derivative pASK75-PA400-Crisantaspase (SEQ ID NO: 14) after seamlessinsertion of a PA#1c/1b(400) (SEQ ID NO: 10) gene cassette via the twoinversely oriented SapI restriction sites. The structural gene for thebiologically/pharmacologically active (pre)proteinPA#1(400)-Crisantaspase (SEQ ID NO: 13) comprising the low repetitivenucleotide sequence encoding a PA#1 polypeptide with 401 amino acidresidues and the structural gene for Crisantaspase as well as codingregion for the bacterial Enx signal sequence (SP^(Enx)) is cloned undertranscriptional control of the tet promoter/operator (tet^(p/o)). Theplasmid backbone outside the expression cassette flanked by the XbaI andHindIII restriction sites is identical with that of the genericexpression vector pASK75 (Skerra (1994) Gene 151:131-135). A plasmid forthe expression of Crisantaspase fused to PA#1(200) (SEQ ID NO: 11) wascloned in the same way using the PA#1b(200) gene cassette (SEQ ID NO:12).

FIG. 6: SDS-PGE analysis of recombinant Crisantaspase genetically fusedwith PA200 or PA400

(A) Analysis of the mature PA#1(400)-Crisantaspase fusion protein (SEQID NO: 13) after periplasmic extraction (PPE), ammonium sulfateprecipitation (ASP) and anion exchange chromatography (AEX) by 10% SDS-PAGE. (B) The gel shows 5 μg samples of purified maturePA#1(200)-Crisantaspase (lane 1) (SEQ ID NO: 11) orPA#1(400)-Crisantaspase (lane 2) (SEQ ID NO: 13). Sizes of the markerproteins (M) are indicated on the left. The PA#1(200)-Crisantaspase andthe PA#1(400)-Crisantaspase fusion protein appear as single homogeneousbands with an apparent molecular size of about 105 kDa (lane 1) or 200kDa (lane 2), respectively. Due to poor SDS binding, PA fusion proteinsgenerally show significantly larger sizes (Schlapschy (2013) Protein EngDes Sel. 26:489-501) than, e.g., the calculated mass of 51 kDa for thePA#1(200)-Crisantaspase monomer or 67 kDa for thePA#1(400)-Crisantaspase monomer.

FIG. 7: Size exclusion chromatography of PASylated Crisantaspasevariants

(A) Overlay of elution profiles for unmodified Crisantaspase, as well asfor Crisantaspase chemically conjugated either to Pga-P/A(20)-Ahx orPga-P/A(40)-Ahx peptides (described in Examples 3 and 2, respectively)and the recombinant Crisantaspase genetically fused with eitherPA#1(200) (SEQ ID NO: 7) or PA#1(400) (SEQ ID NO: 9) polypeptides(described in Example 5). 150 μL of the purified protein at aconcentration of 1 mg/ml was applied to a SUPERDEX S200 10/300 GL columnequilibrated with PBS buffer. Absorption at 280 nm was monitored and thepeak of each chromatography run was normalized to 100%.

(B) Calibration curve for the chromatograms from (A) using a SUPERDEXS200 10/300 GL column. The logarithm of the molecular weight of markerproteins (ovalbumin, 43.0 kDa; bovine serum albumin, 66.3 kDa; alcoholdehydrogenase, 150 kDa, □-amylase, 200 kDa, apo-ferritin, 440 kDa) wasplotted vs. their elution volumes (black circles) and fitted by astraight line. From the observed elution volumes of the tetramericCrisantaspase, its PA#1 peptide modified proteins and its recombinantPA#1 fusion proteins (black squares) their apparent molecular sizes weredetermined as follows. Crisantaspase, 105 kDa (true mass 140 kDa);Crisantaspase/Pga-P/A(20)-Ahx modified protein, 531 kDa (true mass 228kDa); Crisantaspase/Pga-P/A(40)-Ahx modified protein, 820 kDa (true mass284 kDa); PA200-Crisantaspase, 595 kDa (true mass 205 kDa);PA400-Crisantaspase, 1087 kDa (true mass 269 kDa). These data show thatboth the chemically conjugated P/A peptides and the genetic fusion withthe PA#1 polypeptide confer a much enlarged hydrodynamic volume.

FIG. 8: ESI-MS analysis of PASylated Crisantaspase variants

(A) The raw m/z spectrum obtained by Electrospray Ionisation MassSpectrometry (ESI-MS) of the purified Crisantaspase/Pga-P/A(20)-Ahxmodified protein prepared as described in Example 3 was deconvolutedyielding the mass spectrum (B). The observed mass species couldunambiguously be assigned to Crisantaspase conjugated with 9 to 14peptides (cf. Table 3). Major peaks, however, were observed only forprotein species with 10 to 13 peptides, what corresponds to thedetermination of the peptide coupling ratio by SDS-PAGE (cf. Part B ofFIG. 4). (C) and (E) show raw m/z spectra of the PA200-Crisantaspase andPA400-Crisantaspase fusion proteins prepared in Example 5. Thedeconvoluted mass spectra (D) and (F) revealed masses of 51164.75 Da and67199.17 Da, respectively, which correspond almost perfectly to thecalculated masses of 51163.58 Da.

FIG. 9: Mean (±SD) Plasma concentration versus time profiles following asingle IV bolus dose to Male CD-1 mice

The figures shows plasma asparaginase activity of PA-crisantaspaseconjugates following a single IV bolus dose to male mice.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsreferred to herein are incorporated by reference in their entireties.

In one aspect, the present invention relates to a modified proteincomprising (i) an L-asparaginase and (ii) one or more (poly)peptide(s),wherein the (poly)peptide consists solely of proline and alanine aminoacid residues. In a preferred aspect the invention relates to a modifiedprotein comprising (i) an L-asparaginase having at least 85% identity tothe amino acid sequence of SEQ ID NO: 1 and (ii) one or more(poly)peptide(s), wherein the (poly)peptide consists solely of prolineand alanine amino acid residues.

The present invention relates, inter alia, to a modified proteincomprising (i) an L-asparaginase and (ii) one or more (poly)peptide(s),wherein the (poly)peptide consists solely of proline and alanine aminoacid residues. In some embodiments, said L-asparaginase has at least 85%or 100% identity to the amino acid sequence of SEQ ID NO: 1. Inadditional embodiments, the modified protein has an asparaginase orglutaminase activity higher than that of the unmodified L-asparaginase.In further embodiments, said modified protein has an L-asparaginedepletion activity at least about 20% higher than the unmodifiedL-asparaginase. In yet further embodiments, said L-asparaginase is atetramer.

In some embodiments, the modified protein described herein is a modifiedprotein of said L-asparaginase and a polypeptide, wherein thepolypeptide consists solely of proline and alanine amino acid residues.In some embodiments, said polypeptide consists of about 100 to 600proline and alanine amino acid residues, particularly about 200 to about400 proline and alanine amino acid residues. In further embodiments,said polypeptide consists of a total of about 200 proline and alanineamino acid residues or a total of about 400 proline and alanine aminoacid residues. In additional embodiments, said proline residuesconstitute more than about 10% and less than about 70% of thepolypeptide. In yut additional embodiments, said polypeptide comprises aplurality of amino acid repeats, wherein said repeat consists of prolineand alanine residues and wherein no more than 6 consecutive amino acidresidues are identical. For example, said polypeptide comprises orconsists of the amino acid sequence AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 5)or circular permuted versions or (a) multimers(s) of the sequences as awhole or parts of the sequence. In one aspect, said polypeptidecomprises or consists of an amino acid sequence as shown in SEQ ID NO: 7or 9; said polypeptide comprises or consists of an amino acid sequenceencoded by a nucleic acid having a nucleotide sequence as shown in SEQID NO: 8 or 10; said modified protein comprises or consists of an aminoacid sequence as shown in SEQ ID NO: 11 or 13; or said modified proteincomprises or consists of an amino acid sequence encoded by a nucleicacid having a nucleotide sequence as shown in SEQ ID NO: 12 or 14. Insome embodiments, said polypeptide is a random coil polypeptide. Infurther embodiments, the modified protein is a fusion protein of theL-asparaginase and the polypeptide.

In one aspect, the modified protein is a modified protein ofL-asparaginase and one or more peptide(s), wherein each is independentlya peptide R^(N)—(P/A)-R^(C), (P/A) is an amino acid sequence consistingsolely of proline and alanine amino acid residues, wherein R^(N) is aprotecting group attached to the N-terminal amino group of the aminoacid sequence, and R^(C) is an amino acid residue bound via its aminogroup to the C-terminal carboxy group of the amino acid sequence, eachpeptide is conjugated to the L-asparaginase via an amide linkage formedfrom the carboxy group of the C-terminal amino acid residue R^(C) of thepeptide and a free amino group of the L-asparaginase, and at least oneof the free amino groups, which the peptides are conjugated to, is notan N-terminal α-amino group of the L-asparaginase. In some embodiments,said amino acid sequence consists of a total of between 15 to 45 prolineand alanine amino acid residues. In additional embodiments, said aminoacid sequence consists of 20 or 40 proline and alanine amino acidresidues. In further embodiments, said proline residues constitute morethan about 10% and less than about 70% of the amino acid sequence. Forexample, said amino acid sequence is AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 5)or AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 15). In yetfurther embodiments, R^(N) is pyroglutamoyl or acetyl, and/or R^(C) isε-aminohexanoic acid. In some embodiments, the peptides comprised insaid modified protein adopt a random coil conformation. In additionalembodiments, all of the peptides comprised in said modified protein arethe same. In yet additional embodiments, at least one of the free aminogroups, which the peptides are conjugated to, is an ε-amino group of alysine residue of the L-asparaginase. In further embodiments, the freeamino groups, which the peptides are conjugated to, are selected fromthe group comprising the ε-amino group(s) of any lysine residue(s) ofthe L-asparaginase and the N-terminal α-amino group(s) of theL-asparaginase. In yet further embodiments, the L-asparaginase iscomposed of four subunits, and wherein 9 to 13 peptides as defined inany one of items 15 to 24 are conjugated to each subunit of theL-asparaginase.

In one aspect, the polypeptide or peptide described herein mediates adecreased immunogenicity of said modified protein.

In another aspect, the disclosure is related to a nucleic acid encodingthe modified protein described herein. In some embodiments, said nucleicacid is selected from the group consisting of: (a) the nucleic acidcomprising the nucleotide sequence of SEQ ID NO: 12 or 14; (b) thenucleic acid comprising the nucleotide sequence having at least 85%identity to the nucleotide sequence as defined in (a); and (c) thenucleic acid being degenerate as a result of the genetic code to thenucleotide sequence as defined in (a) or (b).

In another aspect, the disclosure is related to a vector comprising thenucleic acid described herein. In another aspect, the disclosure isrelated to a host cell comprising the nucleic acid and/or the vectordescribed herein. In some embodiments, said host cell is selected fromthe group consisting of Pseudomonas fluorescens and Corynebacteriumglutamicum.

In another aspect, the disclosure is related to a process for thepreparation of a modified protein described herein or of a nucleic aciddescribed herein. In some embodiments, the process comprises culturingthe host cell according to item 33 or 34 and isolating said modifiedprotein from the culture or from said cell.

In another aspect, the disclosure is related to a process of preparing amodified protein as defined in any one of items 17 to 29, the processcomprising: (a) coupling an activated peptide of the formulaR^(N)—(P/A)-R^(C-act), wherein R^(C-act) is a carboxy-activated form ofR^(C), wherein R^(C) and (P/A) are as defined in the modified protein tobe prepared, and wherein R^(N) is a protecting group which is attachedto the N-terminal amino group of (P/A), with L-asparaginase to obtain amodified protein of the L-asparaginase and peptides in which R^(N) is aprotecting group. In some embodiments, the activated carboxy group ofthe amino acid residue R^(C-act) in the activated peptide is an activeester group.

In another aspect, the disclosure is related to a composition comprisingthe modified protein described herein or the modified protein preparedby the process described herein. In some embodiments, the composition isa pharmaceutical composition, optionally further comprising (a)pharmaceutical acceptable carrier(s) or excipient(s).

In another aspect, the modified protein described herein, the modifiedprotein prepared by the process described herein, or the compositiondescribed herein may be used as a medicament. In another aspect, themodified protein described herein, the modified protein prepared by theprocess described herein, or the composition described herein may beused in the treatment of a disease, e.g. a disease treatable byL-asparagine depletion in a patient. In another aspect, the modifiedprotein described herein, the modified protein prepared by the processdescribed herein, or the composition described herein may be used in thetreatment of cancer.

In another aspect, the disclosure is related to a method of treating adisease treatable by L-asparagine depletion in a patient, said methodcomprising administering to said patient an effective amount of themodified protein described herein, the modified protein prepared by theprocess described herein, or the composition described herein. In someembodiments, said disease treatable by L-asparagine depletion is acancer. In another aspect, the disclosure is related to a method oftreating cancer comprising the administration of the modified proteindescribed herein, the modified protein prepared by the process describedherein, or the composition described herein, to a subject.

In some embodiments, said cancer is a non-solid cancer. In additionalembodiments, said non-solid cancer is leukemia or non-Hodgkin'slymphoma. In yet additional embodiments, said leukemia is acutelymphoblastic leukemia (ALL) or acute myeloid leukemia (AML); or themethod according to item 48, wherein said leukemia is acutelymphoblastic leukemia (ALL) or acute myeloid leukemia (AML).

In another aspect, said modified protein described herein elicits alower immunogenic response in said patient compared to the unmodifiedL-asparaginase. In some embodiments, said modified protein has a longerin vivo circulating half-life after a single dose compared to theunmodified L-asparaginase. In additional embodiments, said modifiedprotein has a greater AUC value after a single dose compared to theunmodified L-asparaginase. In yet additional embodiments, said patienthas had a previous hypersensitivity to an E. coli L-asparaginase orPEGylated form thereof. In further embodiments, said patient has had aprevious hypersensitivity to an Erwinia L-asparaginase. In yet furtherembodiments, the treatment comprises intravenous administration of saidmodified protein.

In one aspect, the present invention relates to a modified proteincomprising (i) a recombinant L-asparaginase having at least 85% identityto the amino acid sequence of SEQ ID NO: 1 and (ii) one or more(poly)peptide(s), wherein the (poly)peptide consists solely of prolineand alanine amino acid residues. The explanations and definitions givenherein in relation to the terms “modified protein”, “L-asparaginase”,“(poly)peptide(s)” and the like provided herein apply mutatis mutandis.The term “recombinant L-asparaginase” as used herein refers to arecombinant form of L-asparaginase having at least 85% identity to theamino acid sequence of a native Erwinia L-asparaginase. The term“recombinant” may refer to a recombinantly produced L-asparaginase, e.g.a L-asparaginase produced in a host cell comprising a nucleic acidencoding the L-asparaginase.

The modified proteins further show an enhanced plasma half-life and,thus, a prolonged duration of action as compared to the respectiveunconjugated L-asparaginase. This allows for a reduction of the dosingfrequency and thus the side-effect burden. The invention also providesprocesses of preparing the modified proteins as described herein.

In certain aspects, the invention relates to a modified proteincomprising (i) an L-asparaginase having at least 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the amino acidsequence of SEQ ID NO: 1 and (ii) one or more (poly)peptide(s), whereinthe (poly)peptide(s) consist(s) solely of proline and alanine amino acidresidues. It is understood that the term “consisting solely of prolineand alanine amino acid residues” means that at least one proline residueand at least one alanine residue are present, i.e. both at least oneproline residue and at least one alanine residue must be present. In apreferred aspect, the invention relates to a modified protein comprising(i) a recombinant L-asparaginase having the amino acid sequence of SEQID NO: 1 and (ii) one or more (poly)peptide(s), wherein the(poly)peptide consists solely of proline and alanine amino acidresidues. In one aspect, the L-asparaginase is a tetramer (i.e. theL-asparaginase composed of four subunits or monomers). One exemplarysubunit or monomer has the amino acid sequence of SEQ ID NO: 1.

In one aspect, the (poly)peptide (i.e. polypeptide or peptide) mediatesa decreased immunogenicity of the modified protein described herein,e.g. a decreased immunogenicity of the modified protein as compared tothe unconjugated L-asparaginase.

As shown in the appended examples, the PA#1(200)-Crisantaspase proteinhad 109% and the PA#1(400)-Crisantaspase protein had 118% of enzymeactivity compared to the unmodified Crisantaspase; see Example 5. Thisdemonstrates that the fusion of asparaginases as described herein withpolypeptides does not affect enzymatic activity. Surprisingly, theactivity even increased with the length of the PA-polypeptide.

More generally, the herein provided modified proteins have the same orsubstantially the same (enzymatic) activity compared to unmodifiedasparaginase. The (enzymatic) activity may be assessed by the Nesslerassay. Details of the Nessler assay are provided in the appendedexamples and/or are disclosed in the prior art e.g. Mashburn (1963)Biochem. Biophys. Res. Commun. 12, 50 (incorporated herein by referencein its entirety). Accordingly, in one aspect, the herein providedmodified proteins have the same or substantially the same (enzymatic)activity compared to unmodified asparaginase as assessed by a Nesslerassay. The term “unmodified asparaginase” as used herein refers to anative asparaginase, i.e. an asparaginase that is not modified byfusion/conjugation with (poly)peptides as defined herein.

For example, an “unmodified asparaginase” is an L-asparaginase having atleast 85% identity to the amino acid sequence of SEQ ID NO: 1. In apreferred aspect, an “unmodified asparaginase” is an L-asparaginasehaving the amino acid sequence of SEQ ID NO:1.

In some aspects, the herein provided modified proteins have an(enzymatic) activity higher than that of the unmodified L-asparaginase.The (enzymatic) activity may be assessed by the Nessler assay forexample. Details of the Nessler assay are provided in the appendedexamples and/or are disclosed in the prior art e.g. Mashburn (1963)Biochem. Biophys. Res. Commun. 12, 50 (incorporated herein by referencein its entirety). Accordingly, in one aspect, the herein providedmodified proteins have an (enzymatic) activity higher than that of theunmodified L-asparaginase as assessed by a Nessler assay. The term“unmodified asparaginase” as used herein refers to a nativeasparaginase, i.e. an asparaginase that is not modified byfusion/conjugation with (poly)peptides as defined herein. For example,an “unmodified asparaginase” is an L-asparaginase having at least 85%identity to the amino acid sequence of SEQ ID NO: 1. In a preferredaspect, an “unmodified asparaginase” is an L-asparaginase having theamino acid sequence of SEQ ID NO: 1. For example, the modified proteinshave an (enzymatic) activity that can be at least 5% and/or up to 30%(e.g. at least 10%, 15%, 20%, 25% (or more)) higher than that of theL-asparaginase, particularly higher than that of the unmodifiedL-asparaginase, particularly as assessed by the Nessler assay. The aboveexplanations apply in particular to the herein provided fusion proteins(e.g. modified protein of L-asparaginase and a polypeptide, wherein thepolypeptide consists solely of proline and alanine amino acid residues),but are not limited thereto.

In some aspects, the modified proteins have an asparaginase activity orglutaminase activity higher than that of the unmodified L-asparaginase.For example, the modified proteins can have an asparaginase activity orglutaminase activity at least 5% and/or up to 30% (e.g. at least 10%,15%, 20%, 25% (or more)) higher than that of the L-asparaginase,particularly higher than that of the unmodified L-asparaginase. In someembodiments, the asparaginase activity or glutaminase activity may bemeasured by Nessler assay. The rate of hydrolysis of asparagine may bedetermined by measuring released ammonia, and the amount of releasedammonia from using the modified proteins disclosed herein may becompared with that from using the L-asparaginase or unmodifiedL-asparaginase. In additional aspects, said modified proteins have anL-asparagine depletion activity higher than that of the unmodifiedL-asparaginase. In additional embodiments, the modified proteins have anL-asparagine depletion activity at least 5% and/or up to 30% (e.g. atleast 10%, 15%, 20%, 25% (or more)) higher than that of theL-asparaginase, particularly higher than that of the unmodifiedL-asparaginase, for example as assessed by the Nessler assay. Theinvention also relates to a pharmaceutical composition comprising themodified protein, and the modified protein or the pharmaceuticalcomposition for use in therapy, or for use as a medicament, or for usein medicine.

Generally, a modified protein can be obtained by chemical coupling or bygenetic fusion (in the case of conjugation with another protein orpeptide). The term “fusion protein” as used herein relates primarily toa modified protein comprising (i) an L-asparaginase and (ii) one or morepolypeptide(s), wherein the polypeptide consists solely of proline andalanine amino acid residues. In this context, the polypeptide canconsist of about 200 to about 400 proline and alanine amino acidresidues. Exemplary amino acid sequences of such polypeptides are shownin SEQ ID NO: 7 or 9.

If the modified protein is obtained by chemical coupling, it comprises(i) an L-asparaginase and (ii) one or more peptide(s), wherein thepeptide consists solely of proline and alanine amino acid residues. Inthis context, the peptide can consist of a total of between 10 to 100proline and alanine amino acid residues, from about 15 to about 60proline and alanine amino acid residues, from about 15 to 45 proline andalanine amino acid residues, e.g. from about 20 to about 40, forexample, 20 proline and alanine amino acid residues or 40 proline andalanine amino acid residues. Exemplary amino acid sequence of suchpeptides are AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 5) orAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 15).

The term “modified protein” as used herein can be used interchangeablywith the term “conjugate”, particularly if the term “modified protein”refers to a modified protein obtained by chemical coupling or as afusion protein, i.e. primarily if it comprises (i) an L-asparaginase and(ii) one or more (poly)peptide(s), wherein the (poly)peptide consistssolely of proline and alanine amino acid residues. Likewise, the terms“unmodified” and “unconjugated” can be used interchangeably herein.

The invention also relates to a process of preparing the modifiedprotein, comprising (a) coupling an activated peptide of the formulaR^(N)—(P/A)-R^(C-act), wherein R^(C-act) is a carboxy-activated form ofR^(C), wherein R^(C) and (P/A) are as defined in the modified protein tobe prepared, and wherein R^(N) is a protecting group which is attachedto the N-terminal amino group of (P/A), with L-asparaginase to obtain amodified protein of the L-asparaginase and peptides in which R^(N) is aprotecting group.

It has been demonstrated in the appended examples (cf. Example 1,Table 1) that the modified protein can be prepared using a variety ofmass ratios of the activated peptide and asparaginase. For example, massratios of 10:1 (activated peptide: asparaginase), 7.5:1, 5:1 or 3.5:1can be used. It was observed that (enzymatic) activity of the modifiedprotein was highest, if a ratio of 5:1 or below was used (cf. Example 1,Table 2). Thus, it may be advantageous to use a mass ratio of activatedpeptide:asparaginase of 5:1 or below, e.g. 5:1, 4:1, 3.5:1 or 3:1, inthe process described herein above. The term “mass ratio” as used hereinrefers to the ratio of the molecular weight of the activated peptide asdefined herein and of the asparaginase as defined herein (e.g.asparaginase as shown in SEQ ID NO: 1 and proteins with at least 85%identity to SEQ ID NO: 1). The “molecular weight” is typically indicatedherein using the scientific unit Dalton (Da). It is well known that themolecular weight unit of the asparaginase or peptide as indicated hereinin dalton (Da), is an alternative name for the unified atomic mass unit(u). A molecular weight of, e.g., 500 Da is thus equivalent to 500g/mol. The term “kDa” (kilodalton) refers to 1000 Da.

The molecular weight of asparaginase or peptide can be determined usingmethods known in the art, such as, e.g., mass spectrometry (e.g.,electrospray ionization mass spectrometry, ESI-MS, or matrix-assistedlaser desorption/ionization mass spectrometry, MALDI-MS), gelelectrophoresis (e.g., polyacrylamide gel electrophoresis using sodiumdodecyl sulfate, SDS-PAGE), hydrodynamic methods (e.g., gelfiltration/size exclusion chromatography, SEC, or gradientsedimentation), or dynamic (DLS) or static light scattering (e.g.,multi-angle light scattering, MALS), or the molecular weight of theasparaginase or peptide can be calculated from the known amino acidsequence (and the known post-translational modifications, if present) ofthe asparaginase or peptide. Preferably, the molecular weight of theasparaginase or peptide is determined using mass spectrometry.

The invention also relates to a process for the preparation of themodified protein or of a nucleic acid encoding the modified protein. Insome aspects, the process comprises producing an L-asparaginase in ahost selected from the group comprising yeasts, such as Saccharomycescerevisiae and Pichia Pistoris, as well as bacteria, actinomycetes,fungi, algae, and other microorganisms, including Escherichia coli,Bacillus sp., Pseudomonas fluorescens, Corynebacterium glutamicum andbacterial hosts of the following genuses, Serratia, Proteus,Acinetobacter and Alcaligenes. Other hosts are known to those of skillin the art, including Nocardiopsis alba, which expresses a variant ofAsparaginase lacking on glutaminase-activity (Meena et al. (2014)Bioprocess Biosyst. Eng. October 2014 Article, which is incorporated byreference herein in its entirety), and those disclosed in Savitri et al.(2003) Indian Journal of Biotechnology, 2, 184-194, which isincorporated by reference herein in its entirety.

The modified protein can be a fusion protein comprising (i) aL-asparaginase having at least 85% identity to the amino acid sequenceof SEQ ID NO: 1 and (ii) one or more polypeptide(s), wherein thepolypeptide consists solely of proline and alanine amino acid residues.

The proline residues in the polypeptide consisting solely of proline andalanine amino acid residues may constitute more than about 10% and lessthan about 70% of the polypeptide. Accordingly, it is preferred that 10%to 70% of the total number of amino acid residues in the polypeptide areproline residues; more preferably, 20% to 50% of the total number ofamino acid residues comprised in the polypeptide are proline residues;and even more preferably, 30% to 40% (e.g., 30%, 35% or 40%) of thetotal number of amino acid residues comprised in the polypeptide areproline residues.

The polypeptide may comprise a plurality of amino acid repeats, whereinsaid repeat consists of proline and alanine residues and wherein no morethan 6 consecutive amino acid residues are identical. Particularly, thepolypeptide may comprise or consist of the amino acid sequenceAAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 5) or circular permuted versions or (a)multimers(s) of the sequences as a whole or parts of the sequence.

Preferably, the polypeptide comprises or consists of the amino acidsequence as shown in SEQ ID NO: 7 or 9, or the polypeptide comprises orconsists of an amino acid sequence encoded by a nucleic acid having anucleotide sequence as shown in SEQ ID NO: 8 or 10. It is preferredherein that the modified protein (a) comprises or consists of an aminoacid sequence as shown in SEQ ID NO: 11 or 13; or (b) comprises orconsists of an amino acid sequence encoded by a nucleic acid having anucleotide sequence as shown in SEQ ID NO: 12 or 14. In one aspect, thepolypeptide is a random coil polypeptide.

In some aspects, the modified protein, e.g. the fusion protein, has anasparaginase or glutaminase activity higher than that of theunconjugated L-asparaginase. For example, the modified proteins can havean asparaginase or glutaminase activity at least 5% and/or up to 30%(e.g. at least 10%, 15%, 20%, 25% (or more)) higher than that of theunmodified L-asparaginase, particularly as assessed by the Nesslerassay. In further aspects, the L-asparaginase in the modified protein,e.g. in the fusion protein, is covalently linked to a terminal residueof the polypeptide directly by an amine bond, and/or the fusion proteinis manufactured recombinantly. In preferred aspects, the modifiedprotein, e.g. the fusion protein, includes a linker between theL-asparaginase and the polypeptide. An exemplary linker may be analanine amino acid residue. The invention also relates to apharmaceutical composition comprising the modified protein, e.g. thefusion protein, or its use in therapy, or for use as a medicament, orfor use in medicine.

The invention also relates to a nucleic acid encoding the modifiedprotein, particularly a fusion protein as defined herein. Preferably,the nucleic acid is selected from the group consisting of: (a) thenucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:12 or 14; (b) the nucleic acid molecule comprising the nucleotidesequence having at least 85% identity to the nucleotide sequence asdefined in (a); and (c) the nucleic acid molecule being degenerate as aresult of the genetic code to the nucleotide sequence as defined in (a).

One aspect of the invention further relates to a process for thepreparation of a modified protein as defined herein or of a nucleic acidas defined herein. The process may comprise culturing a host cell asdefined herein and isolating said modified protein from the culture orfrom said cell. The process of preparing the modified protein as definedherein, particularly the fusion protein, can comprise culturing a hostcell transformed with or comprising a vector comprising a nucleic acidencoding the modified protein, particularly the fusion protein, underconditions causing expression of the modified protein, particularly ofthe fusion protein. In some aspects, the host cell is selected from thegroup recited above.

The invention further relates to a method of treating a diseasetreatable by L-asparagine depletion in a patient, said method comprisingadministering to said patient an effective amount of the modifiedprotein as defined herein, e.g. the fusion protein. The diseasetreatable by L-asparagine depletion may be a cancer. The modifiedprotein as defined herein may elicit a lower immunogenic response in thepatient compared to unconjugated L-asparaginase, may have a longer invivo circulating half-life after a single dose compared to theunconjugated L-asparaginase, and/or may have a greater AUC value after asingle dose compared to the L-asparaginase (particularly theunconjugated L-asparaginase).

The problem to be solved by the invention can be seen to be theprovision of an L-asparaginase preparation with: high in vitrobioactivity; a stable protein-modifier linkage; prolonged in vivohalf-life; significantly reduced immunogenicity, as evidenced, forexample, by the reduction or elimination of an antibody response againstthe L-asparaginase preparation following repeated administrations;and/or usefulness as a second-line therapy for patients who havedeveloped sensitivity to first-line therapies using, e.g., non-E.coli-derived L-asparaginases.

This problem is solved according to the present invention by theembodiments characterized in the claims, in particular by providing amodified protein comprising an L-asparaginase and a modifier, i.e. (ii)one or more (poly)peptide(s), wherein the (poly)peptide consists solelyof proline and alanine amino acid residues, and by providing methods forpreparing and using the same.

In one aspect, described herein is a modified L-asparaginase withimproved pharmacological properties as compared with the unmodifiedL-asparaginase protein.

The term “modified L-asparaginase” as used herein refers to “a modifiedprotein comprising (i) L-asparaginase and (ii) one or more(poly)peptide(s), wherein the (poly)peptide consists solely of prolineand alanine amino acid residues” as defined and described herein. In oneaspect of the invention the L-asparaginase is derived from Erwiniahaving at least 85% identity to the amino acid of SEQ ID NO: 1.

The modified L-asparaginase described herein, e.g., L-asparaginaseconjugated or fused to one or more (poly)peptide(s), wherein the(poly)peptide consists solely of proline and alanine amino acidresidues, serves as a therapeutic agent particularly for use in patientswho show hypersensitivity (e.g., an allergic reaction or silenthypersensitivity) to treatment with L-asparaginase or PEGylatedL-asparaginase from Erwinia and/or E. coli, or unmodified L-asparaginasefrom Erwinia. The modified L-asparaginase described herein is alsouseful as a therapeutic agent for use in patients who have had a diseaserelapse, e.g., a relapse of ALL, and have been previously treated withanother form of asparaginase.

Erwinia chrysanthemi (also known as Pectobacterium chrysanthemi) hasbeen renamed Dickeya chrysanthemi. Thus, the terms Erwinia chrysanthemi,Pectobacterium chrysanthemi and Dickeya chrysanthemi are usedinterchangeably herein.

Unless otherwise expressly defined, the terms used herein will beunderstood according to their ordinary meaning in the art.

As used herein, the term “including” means “including, withoutlimitation,” and terms used in the singular shall include the plural,and vice versa, unless the context dictates otherwise.

As used herein, the terms “comprising”, “including”, “having” orgrammatical variants thereof are to be taken as specifying the statedfeatures, integers, steps or components but do not preclude the additionof one or more additional features, integers, steps, components orgroups thereof. The terms “comprising”/“including”/“having” encompassthe terms “consisting of” and “consisting essentially of”. Thus,whenever the terms “comprising”/“including”/“having” are used herein,they can be replaced by “consisting essentially of” or, preferably, by“consisting of”.

The terms “comprising”/“including”/“having” mean that any furthercomponent (or likewise features, integers, steps and the like) can bepresent.

The term “consisting of” means that no further component (or likewisefeatures, integers, steps and the like) can be present.

The term “consisting essentially of” or grammatical variants thereofwhen used herein are to be taken as specifying the stated features,integers, steps or components but do not preclude the addition of one ormore additional features, integers, steps, components or groups thereofbut only if the additional features, integers, steps, components orgroups thereof do not materially alter the basic and novelcharacteristics of the claimed product, composition, device or methodand the like.

Thus, the term “consisting essentially of” means that specific furthercomponents (or likewise features, integers, steps and the like) can bepresent, namely those not materially affecting the essentialcharacteristics of the product, composition, device or method. In otherwords, the term “consisting essentially of” (which can beinterchangeably used herein with the term “comprising substantially”),allows the presence of other components in the product, composition,device or method in addition to the mandatory components (or likewisefeatures, integers, steps and the like), provided that the essentialcharacteristics of the product, composition, device or method are notmaterially affected by the presence of other components.

As used herein, the term “about” refers to ±10%, unless indicatedotherwise herein.

As used herein, “a” or “an” may mean one or more.

As used herein, the term “disease treatable by depletion of asparagine”refers to a condition or disorder wherein the cells involved in orresponsible for the condition or disorder either lack or have a reducedability to synthesize L-asparagine. Depletion or deprivation ofL-asparagine can be partial or substantially complete (e.g., to levelsthat are undetectable using methods and apparatus that are known in theart).

As used herein, the term “therapeutically effective amount” refers tothe amount of a protein (e.g., asparaginase or modified proteinthereof), required to produce a desired therapeutic effect.

As used herein, the term, “L-asparaginase” is an enzyme withL-asparagine aminohydrolase activity. L-asparaginase's enzymaticactivity may include not only deamidation of asparagine to aspartic acidand ammonia, but also deamidation of glutamine to glutamic acid andammonia. Asparaginases are typically composed of four monomers (althoughsome have been reported with five or six). Each monomer can be about32,000 to about 36,000 daltons.

Many L-asparaginase proteins have been identified in the art, isolatedby known methods from microorganisms. (See, e.g., Savitri and Azmi,Indian J. Biotechnol 2 (2003) 184¬194, incorporated herein by referencein its entirety). The most widely used and commercially availableL-asparaginases are derived from E. coli or from Erwinia chrysanthemi,both of which share 50% or less structural homology.

The following relates to “L-asparaginase” to be used in accordance withthe invention. Within the Erwinia species, typically 75-77% sequenceidentity was reported between Erwinia chrysanthemi and Erwiniacarotovora-derived enzymes, and approximately 90% sequence identity wasfound between different subspecies of Erwinia chrysanthemi (Kotzia(2007), Journal of Biotechnology 127, 657-669, incorporated herein byreference in its entirety). Some representative Erwinia L-asparaginasesinclude, for example, those provided in Table 1 below which disclosespercent sequence identity to Erwinia Chrysanthemi NCPPB 1066:

TABLE 1 Species Accession No. % Identity Erwinia chrysanthemi 3937AAS67028 91% Erwinia chrysanthemi NCPPB 1125 CAA31239 98% Erwiniacarotovora subsp. atroseptica AAS67027 75% Erwinia carotovora AAP9266677%

The sequences of the Erwinia L-asparaginases and the GenBank entries ofTable 1 are herein incorporated by reference. Exemplary L-asparaginasesused in therapy are L-asparaginase isolated from E. coli and fromErwinia, specifically, Erwinia chrysanthemi.

The L-asparaginases may be native enzymes isolated from themicroorganisms. They can also be produced by recombinant enzymetechnologies in producing microorganisms such as E. coli. As examples,the protein used in the modified protein of the invention can be arecombinant protein produced in an E. coli strain, preferably a proteinfrom an Erwinia species, particularly Erwinia chrysanthemi, produced ina recombinant E. coli strain.

Enzymes can be identified by their specific activities. This definitionthus includes all polypeptides that have the defined specific activityalso present in other organisms, more particularly in othermicroorganisms. Often enzymes with similar activities can be identifiedby their grouping to certain families defined as PFAM or COG. PFAM(protein family database of alignments and hidden Markov models;pfam.xfam.org) represents a large collection of protein sequencealignments. Each PFAM makes it possible to visualize multiplealignments, see protein domains, evaluate distribution among organisms,gain access to other databases, and visualize known protein structures.COGs (Clusters of Orthologous Groups of proteins; ncbi.nlm.nih.gov/COG/)are obtained by comparing protein sequences from 43 fully sequencedgenomes representing 30 major phylogenetic lines. Each COG is definedfrom at least three lines, which permits the identification of formerconserved domains.

The means of identifying percentage sequence identity are well known tothose skilled in the art, and include in particular the BLAST programs,which can be used from the website blast.ncbi.olo.nih.gov/Blast.cgi withthe default parameters indicated on that website. The sequences obtainedcan then be exploited (e.g., aligned) using, for example, the programCLUSTALW (ebi.ac.uk/Tools/clustalw2/index.html) with the defaultparameters. Using the references given on GenBank for known genes, thoseskilled in the art are able to determine the equivalent genes in otherorganisms, bacterial strains, yeasts, fungi, mammals, plants, etc. Thisroutine work is advantageously done using consensus sequences that canbe determined by carrying out sequence alignments with genes derivedfrom other microorganisms, and designing degenerate probes to clone thecorresponding gene in another organism.

The person skilled in the art will understand how to select and designproteins retaining substantially their L-asparaginase activity. Oneapproach for the measuring L-asparaginase activity is a Nessler assay asdescribed by Mashburn (1963) Biochem. Biophys. Res. Commun. 12, 50(incorporated herein by reference in its entirety).

In a particular aspect of the modified protein of the invention, theL-asparaginase has at least about 85% homology or sequence identity tothe amino acid sequence of SEQ ID NO: 1, more specifically at leastabout 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% homology or sequence identity to the amino acidsequence of SEQ ID NO:1 as set forth in the attached sequence listing.The terms “homology” and “sequence identity” are used interchangeablyherein.

The term “comprising the sequence of SEQ ID NO:1” (e.g. if theL-asparaginase has 100% homology or sequence identity to the amino acidsequence of SEQ ID NO: 1) means that the amino-acid sequence of theasparaginase may not be strictly limited to SEQ ID NO:1 but may containone, two, three, four, five, six, seven, eight, nine, ten or moreadditional amino-acids. In other words, if the L-asparaginase to be usedherein has 100% homology or sequence identity to the amino acid sequenceof SEQ ID NO: 1, the L-asparaginase can comprise or consist of the aminoacid sequence of SEQ ID NO: 1. The term “comprising” means in thiscontext that the amino acid sequence of the L-asparaginase of SEQ ID NO:1 may contain one, two, three, four, five, six, seven, eight, nine, tenor more additional amino-acids.

In a particular aspect, the protein is the L-asparaginase of Erwiniachrysanthemi comprising or consisting of the sequence of SEQ ID NO: 1.In another aspect, the L-asparaginase is from Erwinia chrysanthemi NCPPB1066 (Genbank Accession No. CAA32884, incorporated herein by referencein its entirety), either with or without signal peptides and/or leadersequences.

Fragments of the L-asparaginase, preferably the L-asparaginase of SEQ IDNO:1, are also comprised within the definition of the L-asparaginaseused in the modified protein of the invention. The term “a fragment ofasparaginase” (e.g. a fragment of the asparaginase of SEQ ID NO: 1)means that the sequence of the asparaginase may include less amino-acidthan in the asparaginases exemplified herein (e.g. the asparaginase ofSEQ ID NO: 1) but still enough amino-acids to confer L-aminohydrolaseactivity. For example, the “fragment of asparaginase” is a fragment thatis/consists of at least about 150 or 200 contiguous amino acids of oneof the asparaginases exemplified herein (e.g. the asparaginase of SEQ IDNO: 1) (e.g. about 150, 160, 170, 180, 190, 200, 210, 220, 230, 240,250, 260, 270, 280, 290, 300, 310, 320, 321, 322, 323, 324, 325, 326contiguous amino acids) and/or wherein said fragment has up to 50 aminoacids deleted from the N-terminus of said asparaginase exemplifiedherein (e.g. the asparaginase of SEQ ID NO: 1) (e.g. up to 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50) and/or has up toup to 75 or 100 amino acids deleted from the C-terminus of saidasparaginase exemplified herein (e.g. the asparaginase of SEQ ID NO: 1)(e.g. up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 70, 75, 80, 85, 90, 95 or 100) and/or has deleted aminoacids at both the N-terminus and the C-terminus of said asparaginaseexemplified herein (e.g. the asparaginase of SEQ ID NO: 1), wherein thetotal number of amino acids deleted can be up to 125 or 150 amino acids.

It is well known in the art that a polypeptide can be modified bysubstitution, insertion, deletion and/or addition of one or moreamino-acids while retaining its enzymatic activity. The term “one ormore amino acids” in this context can refer to one, two, three, four,five, six, seven, eight, nine, ten or more amino acids. For example,substitution of one amino-acid at a given position by a chemicallyequivalent amino-acid that does not affect the functional properties ofa protein is common Substitutions may be defined as exchanges within oneof the following groups:

-   -   Small aliphatic, non-polar or slightly polar residues: Ala, Ser,        Thr, Pro, Gly    -   Polar, negatively charged residues and their amides: Asp, Asn,        Glu, Gln    -   Polar, positively charged residues: His, Arg, Lys    -   Large aliphatic, non-polar residues: Met, Leu, Ile, Val, Cys    -   Large aromatic residues: Phe, Tyr, Trp.

Thus, changes that result in the substitution of one negatively chargedresidue for another (such as glutamic acid for aspartic acid) or onepositively charged residue for another (such as lysine for arginine) canbe expected to produce a functionally equivalent product.

The positions where the amino-acids are modified and the number ofamino-acids subject to modification in the amino-acid sequence are notparticularly limited. The skilled artisan is able to recognize themodifications that can be introduced without affecting the activity ofthe protein. For example, modifications in the N- or C-terminal portionof a protein may be expected not to alter the activity of a proteinunder certain circumstances. With respect to asparaginases, inparticular, much characterization has been done, particularly withrespect to the sequences, structures, and the residues forming theactive catalytic site. This provides guidance with respect to residuesthat can be modified without affecting the activity of the enzyme. Allknown L-asparaginases from bacterial sources have common structuralfeatures. All are homotetramers with four active sites between the N-and C-terminal domains of two adjacent monomers (Aghaipour (2001)Biochemistry 40, 5655-5664, incorporated herein by reference in itsentirety). All have a high degree of similarity in their tertiary andquaternary structures (Papageorgiou (2008) FEBS J. 275, 4306-4316,incorporated herein by reference in its entirety). The sequences of thecatalytic sites of L-asparaginases are highly conserved between Erwiniachrysanthemi, Erwinia carotovora, and E. coli L-asparaginase II (Id).The active site flexible loop contains amino acid residues 14-33, andstructural analysis show that Thr15, Thr95, Ser62, Glu63, Asp96, andA1a120 contact the ligand (Id). Aghaipour et al. have conducted adetailed analysis of the four active sites of Erwinia chrysanthemiL-asparaginase by examining high resolution crystal structures of theenzyme complexed with its substrates (Aghaipour (2001) Biochemistry 40,5655-5664). Kotzia et al. provide sequences for L-asparaginases fromseveral species and subspecies of Erwinia and, even though the proteinshave only about 75-77% identity between Erwinia chrysanthemi and Erwiniacarotovora, they each still have L-asparaginase activity (Kotzia (2007)J. Biotechnol. 127, 657-669). Moola et al performed epitope mappingstudies of Erwinia chrysanthemi 3937 L-asparaginase and were able toretain enzyme activity even after mutating various antigenic sequencesin an attempt to reduce immunogenicity of the asparaginase (Moola (1994)Biochem. J. 302, 921-927). In view of the extensive characterizationthat has been performed on L-asparaginases, one of skill in the artcould determine how to make fragments and/or sequence substitutionswhile still retaining enzyme activity.

As used herein, the term “about” modifying, for example, the dimensions,volumes, quantity of an ingredient in a composition, concentrations,process temperature, process time, yields, flow rates, pressures, andlike values, and ranges thereof, refers to variation in the numericalquantity that can occur, for example, through typical measuring andhandling procedures used for making compounds, compositions,concentrates or use formulations; through inadvertent error in theseprocedures; through differences in the manufacture, source, or purity ofstarting materials or ingredients used to carry out the methods; andlike considerations. The term “about” also encompasses amounts thatdiffer due to aging of, for example, a composition, formulation, or cellculture with a particular initial concentration or mixture, and amountsthat differ due to mixing or processing a composition or formulationwith a particular initial concentration or mixture. Whether modified bythe term “about” the claims appended hereto include equivalents to thesequantities. The term “about” further may refer to a range of values thatare similar to the stated reference value. In certain embodiments, theterm “about” refers to a range of values that fall within 10, 9, 8, 7,6, 5, 4, 3, 2, 1 percent or less of the stated reference value.

In the context of the present invention, it has surprisingly been foundthat the chemical conjugation of one or more peptides consisting solelyof proline and alanine amino acid residues via a specific C-terminalamino acid residue (R^(C)) to L-asparaginase allows to provide anL-asparaginase modified protein having a particularly high couplingratio of said peptides per molecule of asparaginase and, thus, aconsiderably reduced immunogenicity and enhanced plasma half-life. Ithas further been found that this novel technique can also be applied toL-asparaginase without impairing its catalytic activity, which greatlyenhances the therapeutic value of the corresponding modified proteinsdescribed herein.

In one aspect, described herein is a modified protein comprising (i) anL-asparaginase having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98,99 or 100% identity to the amino acid sequence of SEQ ID NO: 1 and (ii)one or more peptide(s), wherein the peptide consists solely of prolineand alanine amino acid residues.

In a preferred aspect, the modified protein is a modified protein ofL-asparaginase and one or more peptide(s), wherein each is independentlya peptide R^(N)—(P/A)-R^(C), wherein (P/A) is an amino acid sequenceconsisting solely of proline and alanine amino acid residues, whereinR^(N) is a protecting group attached to the N-terminal amino group ofthe amino acid sequence, and wherein R^(C) is an amino acid residuebound via its amino group to the C-terminal carboxy group of the aminoacid sequence, wherein each peptide is conjugated to the L-asparaginasevia an amide linkage formed from the carboxy group of the C-terminalamino acid residue R^(C) of the peptide and a free amino group of theL-asparaginase, and wherein at least one of the free amino groups, whichthe peptides are conjugated to, is not an N-terminal α-amino group ofthe L-asparaginase.

In some aspect, the monomer of the modified protein has from about 350,400, 450, 500, amino acids to about 550, 600, 650, 700, or 750 aminoacids after modification. In additional aspects, the modified proteinhas from about 350 to about 750 amino acids, or about 500 to about 750amino acids.

Each peptide that is comprised in the modified protein as describedherein is independently a peptide R^(N)—(P/A)-R^(C). Accordingly, foreach of the peptides comprised in a modified protein described herein,the N-terminal protecting group R^(N), the amino acid sequence (P/A),and the C-terminal amino acid residue R^(C) are each independentlyselected from their respective meanings. The two or more peptidescomprised in the modified protein may thus be the same, or they may bedifferent from one another. In one aspect, all of the peptides comprisedin the modified protein are the same.

Furthermore, the peptides comprised in the modified protein preferablyadopt a random coil conformation, particularly when the modified proteinis present in an aqueous environment (e.g., an aqueous solution or anaqueous buffer). The presence of a random coil conformation can bedetermined using methods known in the art, in particular by means ofspectroscopic techniques, such as circular dichroism (CD) spectroscopy.

The moiety (P/A) in the chemically conjugated modified protein, which iscomprised in the peptide R^(N)—(P/A)-R^(C), is an amino acid sequencethat can consist of a total of between 10 to 100 or more proline andalanine amino acid residues, a total of 15 to 60 proline and alanineamino acid residues, a total of 15 to 45 proline and alanine amino acidresidues, e.g. a total of 20 to about 40 proline and alanine amino acidresidues, e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45proline and alanine amino acid residues. In a preferred aspect, saidamino acid sequence consists of about 20 proline and alanine amino acidresidues. In another preferred aspect, said amino acid sequence consistsof about 40 proline and alanine amino acid residues. In the peptideR^(N)—(P/A)-R^(C), the ratio of the number of proline residues comprisedin the moiety (P/A) to the total number of amino acid residues comprisedin (P/A) is preferably ≥10% and ≤70%, more preferably ≥20% and ≤50%, andeven more preferably ≥25% and ≤40%. Accordingly, it is preferred that10% to 70% of the total number of amino acid residues in (P/A) areproline residues; more preferably, 20% to 50% of the total number ofamino acid residues comprised in (P/A) are proline residues; and evenmore preferably, 25% to 40% (e.g., 25%, 30%, 35% or 40%) of the totalnumber of amino acid residues comprised in (P/A) are proline residues.Moreover, it is preferred that (P/A) does not contain any consecutiveproline residues (i.e., that it does not contain any partial sequencePP). In a preferred aspect, (P/A) is the amino acid sequenceAAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 5). In another preferred aspect, (P/A)is the amino acid sequence AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA (SEQID NO: 15).

The group R^(N) in the peptide R^(N)—(P/A)-R^(C) may be a protectinggroup which is attached to the N-terminal amino group, particularly theN-terminal α-amino group, of the amino acid sequence (P/A). It ispreferred that R^(N) is pyroglutamoyl or acetyl.

The group R^(C) in the peptide R^(N)—(P/A)-R^(C) is an amino acidresidue which is bound via its amino group to the C-terminal carboxygroup of (P/A), and which comprises at least two carbon atoms betweenits amino group and its carboxy group. It will be understood that the atleast two carbon atoms between the amino group and the carboxy group ofR^(C) may provide a distance of at least two carbon atoms between theamino group and the carboxy group of R^(C) (which is the case if, e.g.,R^(C) is an ω-amino-C₃₋₁₅ alkanoic acid, such as ε-aminohexanoic acid).It is preferred that R^(C) is ε-aminohexanoic acid.

In one embodiment, the peptide is Pga-AAPAAPAPAAPAAPAPAAPA-Ahx-COOH (SEQID NO: 16) or Pga-AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA-Ahx-COOH (SEQID NO: 17). The term “Pga” is an abbreviation of “pyroglutamoyl” or“pyroglutamic acid”. The term “Ahx” is an abbreviation of“ε-aminohexanoic acid”.

As also demonstrated in the appended examples, it has surprisingly beenfound that the use of a C-terminal amino acid residue R^(C) as definedherein, including in particular ε-aminohexanoic acid, allows to providemodified proteins with an advantageously high coupling ratio of peptidesconsisting solely of proline and alanine amino acid residues permolecule of asparaginase and, thus, an advantageously reducedimmunogenicity and an advantageously enhanced plasma half-life.

In the modified proteins as described herein, each peptideR^(N)—(P/A)-R^(C), can be conjugated to the L-asparaginase via an amidelinkage formed from the carboxy group of the C-terminal amino acidresidue R^(C) of the peptide and a free amino group of theL-asparaginase. A free amino group of the L-asparaginase may be, e.g.,an N-terminal α-amino group or a side-chain amino group of theL-asparaginase (e.g., an ε-amino group of a lysine residue comprised inthe L-asparaginase). If the L-asparaginase is composed of multiplesubunits, e.g. if the L-asparaginase is a tetramer, there may bemultiple N-terminal α-amino groups (i.e., one on each subunit). In oneaspect, 9 to 13 peptides as defined herein (e.g. 9, 11, 12, or 13peptides) can be chemically conjugated to the L-asparaginase (e.g. toeach subunit/monomer of the L-asparaginase).

In accordance with the above, in one aspect at least one of the freeamino groups, which the peptides are chemically conjugated to, is not(i.e., is different from) an N-terminal α-amino group of theL-asparaginase. Accordingly, it is preferred that at least one of thefree amino groups, which the peptides are conjugated to, is a side-chainamino group of the L-asparaginase, and it is particularly preferred thatat least one of the free amino groups, which the peptides are conjugatedto, is an ε-amino group of a lysine residue of the L-asparaginase.

Moreover, it is preferred that the free amino groups, which the peptidesare conjugated to, are selected from the ε-amino group(s) of any lysineresidue(s) of the L-asparaginase, the N-terminal α-amino group(s) of theL-asparaginase or of any subunit(s) of the L-asparaginase, and anycombination thereof. It is particularly preferred that one of the freeamino groups, which the peptides are conjugated to, is an N-terminalα-amino group, while the other one(s) of the free amino groups, whichthe peptides are conjugated to, is/are each an ε-amino group of a lysineresidue of the L-asparaginase. Alternatively, it is preferred that eachof the free amino groups, which the peptides are conjugated to, is anε-amino group of a lysine residue of the L-asparaginase.

The modified proteins as described herein are composed of L-asparaginaseand one or more peptides as defined herein. A corresponding modifiedprotein may, e.g., consist of one L-asparaginase and one, two, three,four, five, six, seven, eight, nine, ten, 15, 20, 25, 30, 35, 40, 45,50, 55 (or more) peptides which are each conjugated to theL-asparaginase. The L-asparaginase may be, e.g., a monomeric protein ora protein composed of multiple subunits, e.g. a tetramer. If theL-asparaginase is a monomeric protein, a corresponding modified proteinmay, e.g., consist of one monomeric L-asparaginase and nine to thirteen(or more) (e.g. 9, 10, 11, 12, or 13), peptides which are eachconjugated to the monomeric L-asparaginase. An exemplary amino acidsequence of a monomeric L-asparaginase is shown in SEQ ID NO: 1. If theL-asparaginase is a protein composed of multiple subunits, e.g. of foursubunits (i.e. if said L-asparaginase is a tetramer), a correspondingmodified protein may, e.g., consist of four L-asparaginase subunits andnine to thirteen (or more) (e.g. 9, 10, 11, 12, or 13), peptides asdefined herein which are each conjugated to each subunit of theL-asparaginase. An exemplary amino acid sequence of a subunit ofL-asparaginase is shown in SEQ ID NO. 1. Likewise, if the L-asparaginaseis a protein composed of multiple subunits, e.g. of four subunits (i.e.if said L-asparaginase is a tetramer), a corresponding modified proteinmay, e.g., consist of four L-asparaginase subunits and 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55 (or more) peptides which are each conjugated to theL-asparaginase tetramer. In one aspect the invention relates to amodified protein having an L-asparaginase and multiple chemicallyattached peptide sequences. In a further aspect the length of thepeptide sequences are from about 10 to about 100, from about 15 to about60 or from about 20 to about 40.

The peptide consisting solely of proline and alanine amino acid residuesmay be covalently linked to one or more amino acids of saidL-asparaginase, such as lysine residues and/or N-terminal residue,and/or the peptide consisting solely of proline and alanine amino acidresidues may be covalently linked to at least from about 40, 50, 60, 70,80 or 90% to about 60, 70, 80, 90 or 100% of the accessible amino groupsincluding amino groups of lysine residues and/or N-terminal residue onthe surface of the L-asparaginase. For example, there may be about 11 to12 lysine residues accessible per L-asparaginase, and about 9 to 12lysines would be conjugated to the peptide consisting solely of prolineand alanine amino acid residues. In further aspects, the peptideconsisting solely of proline and alanine amino acid residues iscovalently linked to from about 20, 30, 40, 50, or 60% to about 30, 40,50, 60, 70, 80 or 90% of total lysine residues of said L-asparaginase.In further embodiments, the peptide consisting solely of proline andalanine amino acid residues is covalently linked to the L-asparaginasevia a linker. Exemplary linkers include linkers disclosed in U.S. PatentApplication Publication No. 2015/0037359, which is herein incorporatedby reference in its entirety.

In addition, said modified protein may have a half-life of at leastabout 5, 10, 12, 15, 24, 36, 48, 60, 72, 84 or 96 hours at a dose ofabout 25 μg protein/kg, and/or a longer in vivo circulating half-lifecompared to the unmodified L-asparaginase. Moreover, said modifiedprotein may have a greater area under the plasma drug concentration-timecurve (AUC) compared to the L-asparaginase.

The modified protein according to the present invention can be preparedusing methods known in the art. In particular, it can be prepared usingthe process described in the following, and/or in accordance with or inanalogy to the procedures described in the examples.

The invention further relates to a process of preparing a modifiedprotein as defined herein, the process comprising: (a) coupling anactivated peptide of the formula R^(N)—(P/A)-R^(C-act), whereinR^(C-act) is a carboxy-activated form of R^(C), wherein R^(C) and (P/A)are as defined in the modified protein to be prepared, and wherein R^(N)is a protecting group which is attached to the N-terminal amino group of(P/A), with L-asparaginase to obtain a modified protein of theL-asparaginase and peptides in which R^(N) is a protecting group.

The carboxy-activated C-terminal amino acid residue R^(C-act), which iscomprised in activated peptide, may be any amino acid residue R^(C), asdescribed and defined herein with respect to the peptide, wherein thecarboxy group of R^(C) is in the form of an activated carboxy group.Preferably, the activated carboxy group of the amino acid residueR^(C-act) in the activated peptide is an active ester group.

If the activated carboxy group of R^(C-act) is an active ester group, itis preferably selected from any one the following active ester groups:

or.

A particularly preferred active ester group is a 1-hydroxybenzotriazole(HOBt) active ester group. Accordingly, it is particularly preferredthat the activated carboxy group of R^(C-act) is a group of thefollowing formula:

The process may additionally comprise, before step (a), a further stepof converting a peptide of the formula R^(N)—(P/A)-R^(C), wherein R^(C)and (P/A) are as defined in the modified protein to be prepared, andwherein R^(N) is a protecting group which is attached to the N-terminalamino group of (P/A), into the activated P/A peptide.

For example, in order to obtain an activated peptide having a1-hydroxybenzotriazole active ester group as the activated carboxy groupof R^(C-act), the step of converting the peptide into the activatedpeptide can be conducted by reacting the peptide with a salt of aphosphonium, uronium or immonium ester of 1-hydroxybenzotriazole (HOBt)in the presence of a base. The salt of the phosphonium, uronium orimmonium derivative of HOBt is preferablyO-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU).

The coupling step (a) and the preceding optional step of converting apeptide into an activated peptide can be conducted, e.g., using any ofthe peptide coupling or amide bond formation procedures described in theliterature, e.g., in any of: El-Faham et al., (2011) Chem Rev. 111(11),6557-6602; Montalbetti et al., (2005) Tetrahedron, 61(46), 10827-10852;Klose et al. (1999) Chem. Commun. 18, 1847-1848; Carpino et al. (1995)J. Am. Chem. Soc. 117(19), 5401-5402); Valeur et al., (2009) Chem. Soc.Rev., 38(2), 606-631; or Hermanson, (2013) Bioconjugate techniques.Third edition. Academic press. Suitable reagents and reaction conditionsfor such procedures are further described in the aforementionedliterature and in the further references cited therein. Additionaldescriptions are found in U.S. Pat. Nos. 8,563,521; 9,260,494; and9,221,882, all of which are incorporated by references herein in theirentirety.

Procedures for removing the protecting groups R^(N), as required in theoptional step (b), are well-known in the art and are described, e.g., inWuts et al., (2012) Greene's Protective Groups in Organic Synthesis.Fourth Edition. John Wiley & Sons, and/or in Isidro-Llobet et al.,(2009) Chem. Rev. 109(6), 2455-2504. The optional step (b) can thus beconducted, e.g., as described for the corresponding protecting groupR^(N) in any of the aforementioned references.

In some aspects, the invention relates to a modified protein comprising(i) an L-asparaginase having at least 85, 90, 91, 92, 93, 94, 95, 96,97, 98, 99 or 100% identity to the amino acid sequence of SEQ ID NO: 1and (ii) and a polypeptide, wherein the polypeptide consists solely ofproline and alanine amino acid residues. In one aspect, the modifiedprotein is a fusion protein. The polypeptide consisting solely ofproline and alanine amino acid residues may have a length of about 200to about 400 proline and alanine amino acid residues. In other words thepolypeptide may consist of about 200 to about 400 proline and alanineamino acid residues. In a preferred aspect, the polypeptide consists ofa total of about 200 (e.g. 201) proline and alanine amino acid residues(i.e. has a length of about 200 (e.g. 201) proline and alanine aminoacid residues) or the polypeptide consists of a total of about 400 (e.g.401) proline and alanine amino acid residues (i.e. has a length of about400 (e.g. 401) proline and alanine amino acid residues). In somepreferred embodiments, the polypeptide comprises or consists of an aminoacid sequence as shown in SEQ ID NO: 7 or 9; or the polypeptidecomprises or consists of an amino acid sequence encoded by a nucleicacid having a nucleotide sequence as shown in SEQ ID NO: 8 or 10. Insome aspects, the modified protein, preferably wherein the modifiedprotein is a fusion protein, and each monomer has from about 350, 400,450, 500, amino acids to about 550, 600, 650, 700, 750 or 1,000 aminoacids including the monomer and the P/A amino acid sequence. Inadditional aspects, the modified protein has from about 350 to about 800amino acids or about 500 to about 750 amino acids.

For example, the polypeptide includes the peptides prepared in U.S. Pat.No. 9,221,882.

In a preferred aspect, the modified protein (a) comprises or consists ofan amino acid sequence as shown in SEQ ID NO: 11 or 13; or (b) comprisesor consists of an amino acid sequence encoded by a nucleic acid having anucleotide sequence as shown in SEQ ID NO: 12 or 14. It is contemplatedherein that the modified protein comprises (a) a protein having an aminoacid sequence as shown in SEQ ID NO: 11 or 13; (b) a protein as definedin (a) wherein one to 65 amino acids are deleted, inserted, added orsubstituted in the asparaginase; (c) a protein encoded by a nucleic acidhaving a nucleotide sequence as shown in SEQ ID NO: 12 or 14; (d) aprotein having an amino acid sequence encoded by a nucleic acidhybridizing under stringent conditions to the complementary strand ofnucleic acid molecules as defined in (c); (e) a protein having at least85% identity to the protein of any one of (a) to (d); and (f) a proteinhaving an amino acid sequence encoded by a nucleic acid being degenerateas a result of the genetic code to the nucleotide sequence of a nucleicacid as defined in (c) or (d).

The modified protein as defined herein may be composed of four subunits,wherein the subunits are selected from the group consisting of (a) aprotein having an amino acid sequence as shown in SEQ ID NO: 1; (b) aprotein as defined in (a) wherein one to 65 amino acids are deleted,inserted, added or substituted in the asparaginase; (c) a proteinencoded by a nucleic acid molecule having a nucleotide sequence as shownin SEQ ID NO: 2; (d) a protein having an amino acid sequence encoded bya nucleic acid hybridizing under stringent conditions to thecomplementary strand of nucleic acid molecules as defined in (c); (e) aprotein having at least 85% identity to the protein of any one of (a) to(d); and (f) a protein having an amino acid sequence encoded by anucleic acid being degenerate as a result of the genetic code to thenucleotide sequence of a nucleic acid as defined in (c) or (d).

The invention relates to a nucleic acid encoding the modified protein asdefined herein, specifically if the modified protein is a modifiedprotein of the L-asparaginase and a polypeptide, wherein the polypeptideconsists solely of proline and alanine amino acid residues. In apreferred aspect, the modified protein is a fusion protein. In apreferred aspect, the nucleic acid is selected from the group consistingof: (a) the nucleic acid comprising the nucleotide sequence of SEQ IDNO: 12 or 14; (b) the nucleic acid comprising the nucleotide sequencehaving at least 85% identity to the nucleotide sequence as defined in(a); and (c) the nucleic acid being degenerate as a result of thegenetic code to the nucleotide sequence as defined in (a).

In a further aspect, the invention relates to a nucleotide sequenceencoding the fusion protein, including a nucleotide sequence having atleast 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to thenucleotide sequence selected from the group consisting of SEQ ID NO: 12or 14. While the encoded polypeptide comprises a repetitive amino acidsequence that may form a random coil, the encoding nucleic acidcomprises preferably a low repetitive nucleotide sequences. In otherwords, the nucleic acid can comprise a nucleotide sequence encoding aPA-rich polypeptide, wherein said coding nucleotide sequence comprisesnucleotide repeats having a maximum length of 14, 15, 16, 17, about 20,about 25, about 30, about 35, about 40, about 45, about 50 or about 55nucleotides. The low repetitive nucleic acid as disclosed herein can beadvantageous compared to highly repetitive nucleic acid molecules. Inparticular, the genetic stability of the low repetitive nucleic acidmolecules to be used herein can be improved.

In some aspects, the nucleotide sequence is a sequence encoding any ofthe modified proteins comprising the L-asparaginase and a polypeptide,wherein the polypeptide consists solely of proline and alanine aminoacid residues, preferably wherein the modified protein is a fusionprotein, described herein, except that one or more amino acid is added,deleted, inserted or substituted, with the proviso that the fusionprotein having this amino acid sequence has L-asparaginase activity.

In additional aspects, the invention relates to a (recombinant) vectorcomprising the nucleotide sequence encoding the modified proteincomprising the L-asparaginase and a polypeptide, wherein the polypeptideconsists solely of proline and alanine amino acid residues, preferablywherein the modified protein is a fusion protein, as described herein,wherein the vector can express the modified protein (e.g. fusionprotein). In further aspects, the invention also relates to a hostcomprising the (recombinant) vector described herein. The host may beyeasts, such as Saccharomyces cerevisiae and Pichia Pistoris, bacteria,actinomycetes, fungi, algae, and other microorganisms, includingEscherichia coli, Bacillus sp., Pseudomonas fluorescens, Corynebacteriumglutamicum and bacterial hosts of the following genuses, Serratia,Proteus, Acinetobacter and Alcaligenes. Other hosts are known to thoseof skill in the art, including Nocardiopsis alba, which expresses avariant of Asparaginase lacking on glutaminase-activity (Meena et al.(2014) Bioprocess Biosyst. Eng. October 2014 Article, which isincorporated by reference herein in its entirety), and those disclosedin Savitri et al. (2003) Indian Journal of Biotechnology, 2, 184-194,which is incorporated by reference herein in its entirety.

The present invention relates to a vector comprising the nucleic acid asdescribed herein above, i.e. a nucleic acid encoding the modifiedprotein as defined herein, particularly a modified protein of theL-asparaginase and a polypeptide, wherein the polypeptide consistssolely of proline and alanine amino acid residue, such as a fusionprotein. In a preferred aspect, the nucleic acid is selected from thegroup consisting of: (a) the nucleic acid comprising the nucleotidesequence of SEQ ID NO: 12 or 14; (b) the nucleic acid comprising thenucleotide sequence having at least 85% identity to the nucleotidesequence as defined in (a); and (c) the nucleic acid being degenerate asa result of the genetic code to the nucleotide sequence as defined in(a).

The invention relates to a host cell comprising the nucleic acid asdefined herein or comprising the vector as defined herein. Example hostsare listed above.

The invention further relates to a process of preparing the modifiedprotein as described herein, preferably the fusion protein, or of thenucleic acid encoding same. The process can comprise culturing a hostcell as defined herein and isolating said modified protein from theculture or from said cell. The process can comprise culturing a hostcell (e.g. a host cell transformed with or a host cell comprising thenucleic acid and/or the vector comprising a nucleotide sequence encodingthe modified protein (preferably the fusion protein) under a conditioncausing expression of the modified protein (preferably the fusionprotein). Example hosts are listed above.

Many suitable vectors are known to those skilled in molecular biology.The choice of a suitable vector depends on the function desired,including plasmids, cosmids, viruses, bacteriophages and other vectorsused conventionally in genetic engineering.

Methods which are well known to those skilled in the art can be used toconstruct various plasmids; see, for example, the techniques describedin Sambrook (2012) Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press. Typical plasmid vectors include, e.g., pQE-12,the pUCseries of plasmids, pBluescript (Stratagene), the pET series ofexpression vectors (Novagen) or pCRTOPO (Invitrogen), lambda gt11, pJOE,the pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1. Typicalvectors compatible with expression in mammalian cells include E-027 pCAGKosak-Cherry (L45a) vector system, pREP (Invitrogen), pCEP4(Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5(Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo,pSV2-dhfr, pIZD35, Okayama-Berg cDNA expression vector pcDV1(Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (Invitrogen), pcDNA3.1, pSPORT1(GIBCO BRL), pGEMHE (Promega), pLXIN, pSIR (Clontech), pIRES-EGFP(Clontech), pEAK-10 (Edge Biosystems) pTriEx-Hygro (Novagen) and pCINeo(Promega). Non-limiting examples for plasmid vectors suitable for Pichiapastoris comprise e.g. the plasmids pAO815, pPIC9K and pPIC3.5K (allInvitrogen).

Generally, vectors can contain one or more origins of replication (ori)and inheritance systems for cloning or expression, one or more markersfor selection in the host, e.g., antibiotic resistance, and one or moreexpression cassettes. Examples of suitable origins of replicationinclude, for example, the full length ColE1, its truncated versions suchas those present on the pUC plasmids, the SV40 viral and the M13 phageorigins of replication. Non-limiting examples of selectable markersinclude ampicillin, chloramphenicol, tetracycline, kanamycin, dhfr, gpt,neomycin, hygromycin, blasticidin or geneticin. Further, said vectorcomprises a regulatory sequence that is operably linked to saidnucleotide sequence or the nucleic acid molecule defined herein.

The coding sequence(s), e.g., said nucleotide sequence encoding thepolypeptide, comprised in the vector can be linked to (a)transcriptional regulatory element(s) and/or to other amino acidencoding sequences using established methods. Such regulatory sequencesare well known to those skilled in the art and include, without beinglimiting, regulatory sequences ensuring the initiation of transcription,internal ribosomal entry sites (IRES) and, optionally, regulatoryelements ensuring termination of transcription and stabilization of thetranscript. Non-limiting examples for such regulatory sequences ensuringthe initiation of transcription comprise promoters, a translationinitiation codon, enhancers, insulators and/or regulatory elementsensuring transcription termination. Further examples include Kozaksequences and intervening sequences flanked by donor and acceptor sitesfor RNA splicing, nucleic acid sequences encoding secretion signals or,depending on the expression system used, signal sequences capable ofdirecting the expressed protein to a cellular compartment or to theculture medium.

Examples of suitable promoters include, without being limiting, thecytomegalovirus (CMV) promoter, SV40 promoter, RSV (Rous sarcome virus)promoter, the lacZ promoter, chicken β-actin promoter, CAG promoter (acombination of chicken β-actin promoter and cytomegalovirusimmediate-early enhancer), human elongation factor 1α promoter, AOX1promoter, GAL1 promoter, CaM-kinase promoter, the lac, trp or tacpromoter, the lacUV5 promoter, the T7 or T5 promoter, the Autographacalifornica multiple nuclear polyhedrosis virus (AcMNPV) polyhedralpromoter or a globin intron in mammalian and other animal cells. Oneexample of an enhancer is, e.g., the SV40 enhancer. Non-limitingadditional examples for regulatory elements/sequences ensuringtranscription termination include the SV40 poly-A site, the tk poly-Asite or the AcMNPV polyhedral polyadenylation signals.

Furthermore, depending on the expression system, leader sequencescapable of directing the polypeptide to a cellular compartment orsecreting it into the medium may be added to the coding sequence of thenucleic acid provided herein. The leader sequence(s) is (are) assembledin frame with translation, initiation and termination sequences, andpreferably, a leader sequence is capable of directing secretion oftranslated protein, or a portion thereof, into the periplasmic space orinto the extracellular medium. Suitable leader sequences are, forexample, the signal sequences of BAP (bacterial alkaline phosphatase),CTB (cholera toxin subunit B), DsbA, ENX, OmpA, PhoA, stII, OmpT, PelB,Tat (Twin-arginine translocation) in E. coli, and the signal sequencesof bovine growth hormone, human chymotrypsinogen, human factor VIII,human ig-kappa, human insulin, human interleukin-2, luciferase fromMetrida or Vargula, human trypsinogen-2, inulinase from Kluyveromycesmarxianus, mating factor alpha-1 from Saccharomyces cerevisiae,mellitin, human azurocidin and the like in eukaryotic cells.

The vectors may also contain an additional expressible nucleic acidsequence coding for one or more chaperones to facilitate correct proteinfolding.

In some aspects, the vector of the present invention is an expressionvector. An expression vector is capable of directing the replication andthe expression of the nucleic acid molecule of the invention, e.g., thenucleic acid comprising the nucleotide sequence encoding the polypeptideand the nucleotide sequence encoding asparaginase.

The nucleic acid molecules and/or vectors as described herein above maybe designed for introduction into cells by, e.g., non-chemical methods(electroporation, sonoporation, optical transfection, geneelectrotransfer, hydrodynamic delivery or naturally occurringtransformation upon contacting cells with the nucleic acid molecule ofthe invention), chemical-based methods (calcium phosphate, DMSO, PEG,liposomes, DEAE-dextrane, polyethylenimine, nucleofection etc.),particle-based methods (gene gun, magnetofection, impalefection), phageor phagemid vector-based methods and viral methods. For example,expression vectors derived from viruses such as retroviruses, vacciniavirus, adeno-associated virus, herpes viruses, Semliki Forest Virus orbovine papilloma virus, may be used for delivery of the nucleic acidmolecules into a targeted cell population.

The present invention also relates to a host cell or a non-human hosttransformed with a vector or the nucleic acid described herein. It willbe appreciated that the term “host cell or a non-human host transformedwith the vector” relates to a host cell or a non-human host thatcomprises the vector or the nucleic acid as described herein. Host cellsfor the expression of polypeptides are well known in the art andcomprise prokaryotic cells as well as eukaryotic cells. Appropriateculture media and conditions for the above described host cells areknown in the art.

“Culturing the host or host cell” includes expression of the modifiedprotein, including as a fusion protein, as defined herein and/or thepolypeptide as defined herein and/or of the asparaginase in the host orhost cell.

Methods for the isolation of the modified protein and/or the polypeptideas defined herein and/or of the asparaginase comprise, withoutlimitation, purification steps such as affinity chromatography(preferably using a fusion tag such as the Strep-tag II or theHis₆-tag), gel filtration (size exclusion chromatography), anionexchange chromatography, cation exchange chromatography, hydrophobicinteraction chromatography, high pressure liquid chromatography (HPLC),reversed phase HPLC, ammonium sulfate precipitation orimmunoprecipitation. These methods are well known in the art and havebeen generally described, e.g., in Scopes (1994) ProteinPurification—Principles and Practice, Springer. Such methods providesubstantially pure polypeptides. Said pure polypeptides have ahomogeneity of, preferably, at least about 90 to 95% (on the proteinlevel), more preferably, at least about 98 to 99%. Most preferably,these pure polypeptides are suitable for pharmaceuticaluse/applications.

It is envisaged herein that, a modified protein comprisingL-asparaginase and the polypeptide can be prepared by expressing thenucleic acid molecule comprising the nucleotide sequence encoding thepolypeptide and the nucleic acid sequence encoding the asparaginase. Theexpressed modified protein can be isolated. Alternatively, the modifiedprotein can be prepared by culturing/raising the host comprising thenucleotide sequence or the nucleic acid molecule encoding saidpolypeptide consisting solely of proline and alanine. Thus, the nucleicacid is expressed in the host. The produced polypeptide can be isolated.The produced polypeptide can be conjugated to the asparaginase, e.g.,via a peptide bond or a non-peptide bond.

The modified proteins described herein can be used in the treatment of adisease treatable by depletion of asparagine. The disease treatable bydepletion of asparagines is preferably cancer, such as non-solid cancer.Preferably, the non-solid cancer is leukemia or non-Hodgkin's lymphoma.The leukemia preferably is acute lymphoblastic leukemia (ALL) or acutemyeloid leukemia (AML). For example, the modified proteins are useful inthe treatment or the manufacture of a medicament for use in thetreatment of acute lymphoblastic Leukemia (ALL) in both adults andchildren or acute myeloid leukemia (AML) in both adults and children.The use of the modified proteins described herein in the treatment ofother conditions where asparagine depletion is expected to have a usefuleffect is contemplated. Such conditions include, but are not limited tothe following: malignancies, or cancers, including but not limited tohematalogic malignancies, NK lymphoma, pancreatic cancer, Hodgkin'sdisease, acute myelocytic Leukemia, acute myelomonocytic Leukemia,chronic lymphocytic Leukemia, lymphosarcoma, reticulosarcoma,melanosarcoma, and diffuse large B-cell lymphoma (DLBCL). The cancer maybe a solid cancer, e.g. lung cancer or breast cancer. Representativenon-malignant hematologic diseases which respond to asparagine depletioninclude immune system-mediated Blood diseases, e.g., infectious diseasessuch as those caused by HIV infection (i.e., AIDS). Non-hematologicdiseases associated with asparagine dependence include autoimmunediseases, for example rheumatoid arthritis, SLE, autoimmune, collagenvascular diseases, etc. Other autoimmune diseases includeosteo-arthritis, Issac's syndrome, psoriasis, insulin dependent diabetesmellitus, multiple sclerosis, sclerosing panencephalitis, systemic lupuserythematosus, rheumatic fever, inflammatory bowel disease (e.g.,ulcerative colitis and Crohn's disease), primary billiary cirrhosis,chronic active hepatitis, glomerulonephritis, myasthenia gravis,pemphigus vulgaris, and Graves' disease. Cells suspected of causingdisease can be tested for asparagine dependence in any suitable in vitroor in vivo assay, e.g., an in vitro assay wherein the growth mediumlacks asparagine.

The invention further relates to a method of treating a diseasetreatable by L-asparagine depletion in a patient, said method comprisingadministering to said patient an effective amount of the modifiedprotein. In some preferred aspects, said disease treatable byL-asparagine depletion is Acute Lymphoblastic Leukemia (ALL), acutemyeloid leukemia (AML) or non-Hodgkin's lymphoma. In some aspects, saiddisease treatable by L-asparagine depletion is a cancer including, butnot limited to NK lymphoma, and pancreatic cancer. In additionalembodiments, the modified protein described herein elicits a lowerimmunogenic response in said patient compared to the L-asparaginase ofsaid modified protein.

In some aspects, the modified protein described above has a longer invivo circulating half-life after a single dose compared to theunmodified L-asparaginase of said modified protein. The modified proteindescribed herein can reduce plasma L-asparagine levels for a time periodof at least about 12, 24, 48, 72, 96, or 120 hours when administered ata dose of 5 U/kg body weight (bw) or 10 μg/kg (protein content basis).The modified protein described herein can reduce plasma L-asparaginelevels to undetectable levels for a time period of at least about 12,24, 48, 72, 96, 120, or 144 hours when administered at a dose of 25 U/kgbw or 50 μg/kg (protein content basis). The modified protein describedherein can reduce plasma L-asparagine levels for a time period of atleast about 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, or 240 hourswhen administered at a dose of 50 U/kg bw or 100 μg/kg (protein contentbasis). The modified protein described herein can reduce plasmaL-asparagine levels to undetectable levels for a time period of at leastabout 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, or 240 hours whenadministered at a dose ranging from about 10,000 to about 15,000 IU/m²(about 20-30 mg protein/m²).

The modified protein described herein can result in a similar level ofL-asparagine depletion over a period of time (e.g., 24, 48, or 72 hours)after a single dose.

The modified protein described herein can have a longer t_(1/2) than theunmodified L-asparaginase administered at an equivalent protein dose.The modified protein described above can have a greater AUC value (e.g.at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times) after a single dosecompared to the L-asparaginase of said unmodified protein.

In some aspects the modified protein described herein does not raise anysignificant antibody response for a particular period of time afteradministration of a single dose, e.g., greater than about 1 week, 2weeks, 3 weeks, 4, weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks,10 weeks, 11 weeks, 12 weeks, etc. For example, the modified proteindoes not raise any significant antibody response for at least 8 weeks.In one example, “does not raise any significant antibody response” meansthat the subject receiving the modified protein is identified withinart-recognized parameters as antibody-negative. Antibody levels can bedetermined by methods known in the art, for example ELISA or surfaceplasmon resonance assays (Zalewska-Szewczyk (2009) Clin. Exp. Med. 9,113-116; Avramis (2009) Anticancer Research 29, 299-302, each of whichis incorporated herein by reference in its entirety). The modifiedprotein may have any combination of these properties.

In some aspects, treatment with the modified protein described hereinwill be administered as a first line therapy. In another aspect,treatment with the modified protein will be administered as a secondline therapy in patients, particularly patients with ALL, whereobjective signs of allergy or hypersensitivity, including “silenthypersensitivity,” have developed to other asparaginase preparations, inparticular, the native Escherichia coli-derived L-asparaginase or itsPEGylated variant (pegaspargase). Non-limiting examples of objectivesigns of allergy or hypersensitivity include testing “antibody positive”for an asparaginase enzyme. In a specific aspect, the modified proteinis used in second line therapy after treatment with pegaspargase. Thepatient may have had a previous hypersensitivity to an E. coliL-asparaginase, and/or may have had a previous hypersensitivity to anErwinia L-asparaginase. The hypersensitivity may be selected from thegroup consisting of allergic reaction, anaphylactic shock, and silenthypersensitivity.

The incidence of relapse in ALL patients following treatment withL-asparaginase remains high, with approximately 10-25% of pediatric ALLpatients having early relapse (e.g., some during maintenance phase at30-36 months post-induction) (Avramis (2005) Clin. Pharmacokinet. 44,367-393). If a patient treated with E. coli-derived L-asparaginase has arelapse, subsequent treatment with E. coli preparations could lead to a“vaccination” effect, whereby the E. coli preparation has increasedimmunogenicity during the subsequent administrations. The modifiedprotein described herein may be used in a method of treating patientswith relapsed ALL who were previously treated with other asparaginasepreparations, in particular those who were previously treated with E.coli-derived asparaginases. The disease relapse may occur aftertreatment with an E. coli L-asparaginase or PEGylated form thereof.

In another aspect, the invention is directed to a method for treatingacute lymphoblastic Leukemia comprising administering to a patient inneed of the treatment a therapeutically effective amount of the modifiedprotein described above. In a specific aspect, treatment will beadministered at a dose ranging from about 1500 IU/m² to about 15,000IU/m², typically about 10,000 to about 15,000 IU/m² (about 20-30 mgprotein/m²), at a schedule ranging from about twice a week to about oncea month, typically once per week or once every other week. The modifiedprotein described above may be administered as a single agent (e.g.,monotherapy) or as a part of a combination of chemotherapy drugs,including, but not limited to glucocorticoids, corticosteroids,anticancer compounds or other agents, including, but not limited tomethotrexate, dexamethasone, prednisone, prednisolone, vincristine,cyclophosphamide, and anthracycline. As an example, patients with ALLwill be administered the modified protein described above as a componentof multi-agent chemotherapy during 3 chemotherapy phases includinginduction, consolidation or intensification, and maintenance. In aspecific example, the modified protein described above is notadministered with an asparagine synthetase inhibitor (e.g., such as setforth in WO 2007/103290, which is herein incorporated by reference inits entirety). In another specific example, the modified proteindescribed above is not administered with an asparagine synthetaseinhibitor, but is administered with other chemotherapy drugs. Themodified protein described above can be administered before, after, orsimultaneously with other compounds as part of a multi-agentchemotherapy regimen.

In a specific embodiment, the method comprises administering themodified protein described above at an amount of about 1 U/kg to about25 U/kg (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 U/kg) or an equivalent amountthereof 20 (e.g., on a protein content basis). The amounts of themodified protein to be delivered will depend on many factors, forexample, the IC₅₀, EC₅₀, the biological half-life of the compound, theage, size, weight, and physical condition of the patient, and thedisease or disorder to be treated. The importance of these and otherfactors to be considered are well known to those of ordinary skill inthe art. In certain embodiments, the amount of modified protein to beadministered may range from about 10 International Units per squaremeter of the surface area of the patient's body (IU/m²) to 50,000 IU/m².In additional aspects, the modified protein is administered at an amountselected from the group consisting of about 5, about 10, and about 25U/kg. In another specific aspect, the modified protein is administeredat a dose ranging from about 1,000 IU/m2 to about 20,000 IU/m 2 (e.g.,1,000 IU/m², 2,000 IU/m², 3,000 IU/m², 4,000 IU/m², 5,000 IU/m², 6,000IU/m², 7,000 IU/m², 8,000 IU/m², 9,000 IU/m², 10,000 IU/m², 11,000IU/m², 25 12,000 IU/m², 13,000 IU/m², 14,000 IU/m², 15,000 IU/m², 16,000IU/m², 17,000 IU/m², 18,000 IU/m², 19,000 IU/m², or 20,000 IU/m²). Inanother specific aspect, the modified protein described above isadministered at a dose that depletes L-asparagine to undetectable levelsusing methods and apparatus known in the art for a period of about 3days to about 10 days (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 days) for asingle dose.

The modified protein may be administered in a dose that depletesL-asparagine to undetectable levels for a period of about 3 days toabout 10 days, about 5 days to 20 days, about 1 day to 15 days, or about2 day to 30 days. The modified protein may be administered in a dosethat depletes L-asparagine to undetectable levels for a period of about1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days to about 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20 days. The modified protein may beadministered intravenously or intramuscularly. In additionalembodiments, said modified protein may be administered once or twice perweek, less than once per week, or as monotherapy.

The present invention relates to a composition comprising the modifiedprotein as defined herein or the modified protein prepared by theprocess as described herein. The composition may be a pharmaceuticalcomposition, optionally further comprising (a) pharmaceutical acceptablecarrier(s) or excipient(s).

The invention also relates to a pharmaceutical composition comprisingthe modified protein described above. In a specific aspect, thepharmaceutical composition is contained in a vial as a lyophilizedpowder to be reconstituted with a solvent, such as currently availablenative L-asparaginases, whatever the bacterial source used for itsproduction (e.g. KIDROLASE, ELSPAR, ERWINASE). In another aspect, thepharmaceutical composition is a solution, such as pegaspargase(ONCASPAR) enabling, further to an appropriate handling, anadministration through, e.g., intramuscular, intravenous (infusionand/or bolus), intra-cerebro-ventricular (icy), sub-cutaneous routes.

The modified protein, including compositions comprising the same (e.g.,a pharmaceutical composition) can be administered to a patient usingstandard techniques. Techniques and formulations generally may be foundin Remington's Pharmaceutical Sciences, 22nd ed., Pharmaceutical Press,(2012). Suitable dosage forms, in part, depend upon the use or the routeof entry, for example, oral, transdermal, transmucosal, or by injection(parenteral). Such dosage forms should allow the therapeutic agent toreach a target cell or otherwise have the desired therapeutic effect.For example, pharmaceutical compositions injected into the blood streampreferably are soluble. The pharmaceutical compositions according to theinvention can be formulated as pharmaceutically acceptable salts andcomplexes thereof. Pharmaceutically acceptable salts are non-toxic saltspresent in the amounts and concentrations at which they areadministered. The preparation of such salts can facilitatepharmaceutical use by altering the physical characteristics of thecompound without preventing it from exerting its physiological effect.Useful alterations in physical properties include lowering the meltingpoint to facilitate transmucosal administration and increasingsolubility to facilitate administering higher concentrations of thedrug. The pharmaceutically acceptable salt of a modified protein asdescribed herein may be present as a complex, as those in the art willappreciate. Pharmaceutically acceptable salts include acid additionsalts such as those containing sulfate, hydrochloride, fumarate,maleate, phosphate, sulfamate, acetate, citrate, lactate, tartrate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate,cyclohexylsulfamate, and quinate. Pharmaceutically acceptable salts canbe obtained from acids, including hydrochloric acid, maleic acid,sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid,lactic acid, tartaric acid, malonic acid, methanesulfonic acid,ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid,cyclohexylsulfamic acid, fumaric acid, and quinic acid. Pharmaceuticallyacceptable salts also include basic addition salts such as thosecontaining benzathine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine, procaine, aluminum, calcium, lithium,magnesium, potassium, sodium, ammonium, alkylamine, and zinc, whenacidic functional groups, such as carboxylic acid or phenol are present.For example, see Remington's Pharmaceutical Sciences, supra. Such saltscan be prepared using the appropriate corresponding bases.Pharmaceutically acceptable carriers and/or excipients can also beincorporated into a pharmaceutical composition according to theinvention to facilitate administration of the particular asparaginase.Examples of carriers suitable for use in the practice of the inventioninclude calcium carbonate, calcium phosphate, various sugars such aslactose, glucose, or sucrose, or types of starch, cellulose derivatives,gelatin, vegetable oils, polyethylene glycols, and physiologicallycompatible solvents. Examples of physiologically compatible solventsinclude sterile solutions of water for injection (WFI), saline solutionand dextrose. Pharmaceutical compositions according to the invention canbe administered by different routes, including intravenous,intraperitoneal, subcutaneous, intramuscular, oral, topical(transdermal), or transmucosal administration. For systemicadministration, oral administration is preferred. For oraladministration, for example, the compounds can be formulated intoconventional oral dosage forms such as capsules, tablets, and liquidpreparations such as syrups, elixirs, and concentrated drops.Alternatively, injection (parenteral administration) may be used, e.g.,intramuscular, intravenous, intraperitoneal, and subcutaneous injection.For injection, pharmaceutical compositions are formulated in liquidsolutions, preferably in physiologically compatible buffers orsolutions, such as saline solution, Hank's solution, or Ringer'ssolution. In addition, the compounds may be formulated in solid form andredissolved or suspended immediately prior to use. For example,lyophilized forms of the modified protein can be produced. In a specificaspect, the modified protein is administered intramuscularly. Inpreferred specific aspect, the modified protein is administeredintravenously.

Systemic administration can also be accomplished by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are well known in the art, and include, forexample, for transmucosal administration, bile salts, and fusidic acidderivatives. In addition, detergents may be used to facilitatepermeation. Transmucosal administration, for example, may be throughnasal sprays, inhalers (for pulmonary delivery), rectal suppositories,or vaginal suppositories. For topical administration, compounds can beformulated into ointments, salves, gels, or creams, as is well known inthe art.

In one aspect, the invention also relates to the use of the modifiedprotein as described herein in therapy. The use may be for treating adisease treatable by L-asparagine depletion described above as a methodof treating a disease treatable by L-asparagine depletion. In oneaspect, the invention relates to the modified protein as describedherein or the modified protein prepared by the process as describedherein, or the composition comprising the modified protein as describedherein, for use as a medicament/for use in therapy/for use in medicine.

In one aspect, the invention relates to the modified protein asdescribed herein or the modified protein prepared by the process asdescribed herein, or the composition comprising the modified protein asdescribed herein, for use in the treatment of a disease treatable byL-asparagine depletion in a patient. The present invention also relatesto the use of the modified protein as described herein or of themodified protein prepared by the process as described herein, or of thecomposition comprising the modified protein as described herein in thepreparation of a medicament for treating a disease treatable byL-asparagine depletion in a patient, The present invention also relatesto a method of treating a disease treatable by L-asparagine depletion ina patient, said method comprising administering to said patient aneffective amount of the modified protein as described herein, themodified protein prepared by the process as described herein, orcomposition as described herein. Preferably, the disease treatable byL-asparagine depletion is a cancer.

In a preferred aspect, the invention relates to the modified protein asdescribed herein or the modified protein prepared by the process asdescribed herein, or the composition comprising the modified protein asdescribed herein for use in the treatment of cancer. The presentinvention also relates to the use of the modified protein as describedherein or of the modified protein prepared by the process as describedherein, or of the composition comprising the modified protein asdescribed herein in the preparation of a medicament for treating cancer.The present invention also relates to a method for treating cancercomprising the administration of the modified protein described herein,the modified protein prepared by the process described herein, or thecomposition described herein, to a subject.

It is preferred herein that the subject to be treated is a mammal,particularly a human

The cancer may be a non-solid cancer, e.g. is leukemia or non-Hodgkin'slymphoma. Preferably, said leukemia is acute lymphoblastic leukemia(ALL) or acute myeloid leukemia (AML).

The modified protein may elicit a lower immunogenic response in thepatient compared to the unconjugated L-asparaginase. The modifiedprotein may have a longer in vivo circulating half-life after a singledose compared to the unconjugated L-asparaginase. The modified proteincan have a greater AUC value after a single dose compared to theunconjugated L-asparaginase. The patient may have had a previoushypersensitivity to an E. coli L-asparaginase or PEGylated form thereof.

The following examples illustrate exemplary embodiments of theinvention:

Example 1: Optimization of Coupling Ratio for the Preparation ofPyroglutamoyl-P/A(20)-Aminohexanoyl-Crisantaspase

4.38 mg Pga-P/A#1(20)-Ahx peptide (Part A of FIG. 1; TFA salt, purity98%; PSL Peptide Specialty Laboratories, Heidelberg, Germany) (SEQ IDNO: 16, amino acid sequence shown in SEQ ID NO: 5) was dissolved in 66.3μl DMSO. The chemical activation of the P/A peptide via its terminalcarboxylate group was started by addition of 23.7 μL of a solution of500 mM TBTU (CAS#125700-67-6; Iris Biotech, Marktredwitz, Germany) inDMSO and 2.7 μL DIPEA to the peptide solution and vortexing (cf. Part Cof FIG. 1). In this setup, the concentration of the peptide was 25.8 mMand the molar ratio between DIPEA, TBTU and Pga-P/A#1(20)-Ahx was 5:5:1.After 10 min incubation at 25° C. the mixture was diluted in Eppendorftubes according to Table 1.

A solution of Dickeya chrysanthemi L-Asparaginase (Crisantaspase, SEQ IDNO: 1, recombinant, produced in E. coli (lot RE-LAP-P57D) with aconcentration of 2 mg/mL was prepared in phosphate-buffered saline (PBS:115 mM NaCl, 4 mM KH₂PO₄ and 16 mM Na₂HPO₄, pH 7.4) and pipetted intoeach Eppendorf tube according to the volumes stated in table 1. Aftermixing by repeated pipetting and vortexing, the coupling reaction wasallowed to take place at 25° C. for 30 min. The reaction was quenched byaddition of glycine (pH 8.0 adjusted with Tris base) to a finalconcentration of 250 mM.

TABLE 1 Dilution series of activated P/A peptide for coupling withAsparaginase Peptide stock DMSO Asparaginase Mass ratio solution [μL][μL] [μL] 10x   21 0 50 7.5x 21 7 66.7 5x   21 21 100 3.5x 21 39 143

SDS-PAGE analysis of the modified proteins is shown in FIG. 2. Theindividual bands correspond to protein modified proteins varying by onecoupled P/A peptide each. The additional application of a mix ofcoupling reactions with ratios of 0.3 to 10 mg peptide per mg proteinallowed counting of the bands in a successive ladder starting from theunconjugated protein and thus the number of coupled P/A peptides couldbe precisely determined. The band intensities were quantifieddensitometrically using the Quant v12.2 software (TotalLab, Newcastleupon Tyne, UK) and arithmetic mean values of the number of coupledpeptides per Crisantaspase monomer weighted for their band intensitieswere calculated (cf. Table 2). 3.5 mg P/A peptide per mg Crisantaspaseresulted in a coupling ratio in the range of 9 to 12 P/A peptides perCrisantaspase monomer (mean value: 10.4). Increasing the applied massratio to 10 mg P/A peptide per mg Crisantaspase led only to a slightincrease of the resulting coupling ratio of 10 to 13 P/A peptides perCrisantaspase (mean value 12.0), indicating a saturation of accessibleamino groups.

The modified proteins were purified by anion exchange chromatography(AEX) on a MonoQ HR5/5 column (GE Healthcare) using 25 mM Na-borate pH9.0, 1 mM EDTA as running buffer and a NaCl concentration gradient from0 to 1 M to elute the proteins. L-asparaginase aminohydrolase activityof each Crisantaspase modified protein was determined by reaction ofammonia that is liberated via L-asparagine enzymatic activity with theNessler reagent. Briefly, 50 μL of enzyme solution was mixed with 20 mMof L-asparagine in a 100 mM sodium borate buffer pH 8.6 containing0.015% (w/v) bovine serum albumin and incubated for 15 min at 37° C. Thereaction was stopped by addition of 200 μL of Nessler reagent(Sigma-Aldrich). Absorbance of this solution was measured at 450 nm. Theactivity was calculated from a calibration curve that was obtained fromammonium sulphate as reference. The results are summarized in Table 2.

TABLE 2 Enzymatic activities of Crisantaspase conjugated withPga-P/A(20)-Ahx peptide in different amounts mg PA peptide/ mol PApeptide/ Specific activity Rel. activity mg Crisantaspase mol monomer[U/mg] [%] 0 — 540 ± 32 100 3.5 10.4 508 ± 20 94.1 5 11.2 436 ± 22 80.77.5 11.7 401 ± 21 74.3 10 12.0 256 ± 20 47.4

Example 2: Preparation ofPyroglutamoyl-P/A(40)-Aminohexanoyl-Crisantaspase

28 mg of the Pyroglutamoyl-P/A#1(40)-Ahx peptide (SEQ ID NO. 17, aminoacid sequence shown in SEQ ID NO: 15), Part B of FIG. 1, TFA salt,purity 98%; Almac Group, Craigavon, UK) was dissolved in 1324 μL ofanhydrous DMSO (99.9%; Sigma-Aldrich, Taufkirchen, Germany). To achievechemical activation of the P/A peptide via its terminal carboxylategroup, 162 μL of a solution of 500 mM TBTU (CAS#125700-67-6; IrisBiotech, Marktredwitz, Germany) in DMSO and, after mixing, 14 μL DIPEA(99.5%, biotech. Grade, Sigma-Aldrich) were added. The whole mixture wasvortexed briefly and incubated for 20 min at 25° C. (cf. Part C of FIG.1). In this setup, the peptide concentration was 5.41 mM and the molarratio between DIPEA, TBTU and Pga-P/A#1(40)-Ahx was 10:10:1.

3.5 mL of an ice-cold Crisantaspase solution (SEQ ID NO: 1) (2 mg/mL inPBS) was mixed with the activated peptide solution (1.5 mL), resultingin a mass ratio between Pga-P/A#1(40)-Ahx and Crisantaspase of 5:1, andincubated at room temperature for 30 min to allow coupling. Using aregenerated cellulose membrane dialysis tube (MWCO 50 kDa, SpectrumLaboratories, Los Angeles, Calif.), the solution was dialyzed against 5L AEX running buffer (25 mM Na-borate pH 9.0, 1 mM EDTA) and subjectedto anion exchange chromatography on a HISCALE 16/40 column packed withSOURCE 15Q resin (GE Healthcare). The column was equilibrated with AEXrunning buffer and the protein modified protein was eluted using asegmented NaCl concentration gradient from 0 to 150 mM in 1 columnvolume and from 150 to 1000 mM in 0.25 column volumes (Part A of FIG.3).

Applying the eluate to SDS-PAGE alongside a ladder obtained from a mixof coupling reactions with ratios of 0.3 to 10 mg peptide per mg proteinallowed determination of the coupling ratio of 9-11 PA peptides perCrisantaspase monomer (mean value: 10.0) (Part B of FIG. 3). Enzymeactivity of the Crisantaspase/PA(40) modified protein determined usingthe Nessler assay described in example 1 was 78.2% of the activity ofthe equally assayed non-modified Crisantaspase.

Example 3: Preparation ofPyroglutamoyl-P/a(20)-Aminohexanoyl-Crisantaspase

21 mg of the Pyroglutamoyl-P/A#1(20)-Ahx peptide (SEQ ID NO: 5, Part Aof FIG. 1; TFA salt, purity 98%; PSL Peptide Specialty Laboratories,Heidelberg, Germany) was dissolved in 1376 μL of anhydrous DMSO (99.9%;Sigma-Aldrich, Taufkirchen, Germany). To achieve chemical activation ofthe P/A peptide via its terminal carboxylate group, 114 μL of a solutionof 500 mM TBTU (CAS#125700-67-6; purchased from Iris Biotech,Marktredwitz, Germany) in DMSO and, after mixing, 10 μL DIPEA (99.5%,biotech. Grade, Sigma-Aldrich) were added. The whole mixture wasvortexed briefly and incubated for 20 min at 25° C. (Part C of FIG. 1).In this setup, the peptide concentration was 7.58 mM and the molar ratiobetween DIPEA, TBTU and Pga-P/A#1(20)-Ahx was 5:5:1.

3.5 mL of an ice-cold Crisantaspase solution (SEQ ID NO: 1) (2 mg/mL inPBS) was mixed with the activated peptide solution (1.5 mL), resultingin a mass ratio between Pga-P/A#1(40)-Ahx and Crisantaspase of 5:1, andincubated at room temperature for 30 min to allow coupling. Using aregenerated cellulose membrane dialysis tube (MWCO 50 kDa, SpectrumLaboratories, Los Angeles, Calif.), the solution was dialyzed against 5L AEX running buffer (25 mM Na-borate pH 9.0, 1 mM EDTA) and subjectedto anion exchange chromatography on a HISCALE 16/40 column packed withSOURCE 15Q resin (GE Healthcare). The column was equilibrated with AEXrunning buffer and the protein modified protein was eluted using asegmented NaCl concentration gradient from 0 to 150 mM in 1 columnvolume and from 150 to 1000 mM in 0.25 column volumes (Part A of FIG.4).

Applying the eluate to SDS-PAGE alongside a ladder obtained from a mixof coupling reactions with ratios of 0.3 to 10 mg peptide per mgprotein, allowed determination of the coupling ratio of 10-13 PApeptides per Crisantaspase monomer (mean value 11.9) (Part B of FIG. 4).Enzyme activity of the Crisantaspase/PA(20) modified protein determinedusing the Nessler assay described in Example 1 was 91.2% of the activityof the equally assayed non-modified Crisantaspase.

Example 4: Cloning of Expression Plasmids for the Periplasmic Productionof Crisantaspase N-Terminally Fused to P/A Sequences of Varying Length

A synthetic DNA fragment encoding the mature amino acid sequence ofDickeya chrysanthemi L-asparaginase (UniProt ID P06608) was obtainedfrom a gene synthesis provider (Thermo Fisher Scientific, Regensburg,Germany). This gene fragment (SEQ ID NO: 4) comprised an XbaIrestriction site, followed by a ribosomal binding site, the nucleotidesequence encoding the Enx signal peptide, followed by a GCC alaninecodon, a first SapI recognition sequence GCTCTTC on the non-codingstrand, an 11-nucleotide spacer, and a second SapI restriction sequencein reverse complementary orientation, with its recognition sequenceGCTCTTC on the coding strand, followed by a GCC alanine codon directlylinked to the coding sequence for mature L-asparaginase, which wasfinally followed by a HindIII restriction site.

This gene fragment was cloned on pASk75 via the flanking restrictionsites XbaI and HindIII according to standard procedures (Sambrook (2012)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress). The resulting plasmid (Part A of FIG. 5) was digested with SapI,which led to the liberation of a small (30 bp) DNA insert containingboth SapI recognition sites and a cleaved vector backbone withcompatible 5′-GCC/5′-GGC sticky ends at the position directly in frontof the encoded mature N-terminus of L-asparaginase, which is ideallysuited for insertion of the low repetitive nucleic acid moleculeencoding the proline/alanine-rich amino acid repeat sequence. Afterisolation of the vector fragment using the Promega Wizard gel extractionkit (Promega, Mannheim, Germany) and dephosphorylation with thethermosensitive alkaline phosphatase FastAP (Thermo Fisher Scientific,Waltham, Mass.), both according to the manufacturer's instructions, itwas ligated with the PA#1b(200) gene cassette excised frompXL2-PA#1b(200) (SEQ ID NO: 8) or PA#1c/1b(400) gene cassette excisedfrom pXL2-PA#1c/1b(400) (SEQ ID NO: 10) via Earl restriction digest. Theresulting plasmids (SEQ ID NO: 12 and SEQ ID NO: 14)(Part B of FIG. 5)allow the bacterial expression of fusion proteins (SEQ ID NO: 11 and SEQID NO: 13) consisting of a proline/alanine-rich amino acid repeatsequence fused with the biologically active protein Crisantaspase (afterin vivo processing of the Enx signal peptide upon periplasmic secretionin E. coli).

Example 5: Bacterial Production and Purification of Fusion ProteinsBetween Either the PA#1(200) or the PA#1(400) Sequence and Crisantaspase

Both, the PA#1(200)-Crisantaspase and the PA#1(400)-Crisantaspase fusionprotein (calculated mass: 51 kDa and 67 kDa, respectively) were producedat 25° C. in E. coli W3110 harboring the expression plasmidpASK75-PA200-Crisantaspase or pASK75-PA400-Crisantaspase (Part B of FIG.5) from Example 4 using an 8 L bench top fermenter with a syntheticglucose mineral medium supplemented with 100 mg/L ampicillin accordingto a published procedure (Schiweck (1995) Proteins 23: 561-565).Recombinant gene expression was induced by addition of 500 μg/Lanhydrotetracycline (Skerra (1994) loc. cit.) as soon as the culturereached OD₅₅₀=40. After an induction period of 2.5 h, cells wereharvested by centrifugation and resuspended during 10 min in ice-coldperiplasmic fractionation buffer (500 mM sucrose, 1 mM EDTA, 200 mMboric acid/NaOH pH 8.0; 2 ml per L and OD₅₅₀). After adding 15 mM EDTAand 250 μg/mL lysozyme, the cell suspension was incubated for 20 min onice, centrifuged several times, and the cleared supernatant containingthe recombinant protein was recovered.

The periplasmic extracts were dialyzed twice at 4° C. against 15 L PBScontaining 1 mM EDTA for at least 6 h, respectively, filtered using a0.2 μm cellulose nitrate membrane (GE Healthcare) and precipitated byaddition of ammonium sulfate (Ph. Eur. grade; Applichem, Darmstadt,Germany) to a saturation of 25% at 25° C. After centrifugation, thesupernatant was removed and the sediment was resuspended in AEX runningbuffer (25 mM Na-borate pH 9.0, 1 mM EDTA) and dialyzed at 4° C. against5 L AEX running buffer for at least 6 h. The dialyzed protein solutionwas cleared from remaining insoluble matter by centrifugation andsubjected to subtractive anion exchange chromatography using a 85 mlHISCALE column (GE Healthcare, Freiburg, Germany) packed with Source 15Qresin, connected to an ÄKTA purifier system (GE Healthcare, Freiburg,Germany), equilibrated in AEX running buffer. The column flow-throughcontaining the pure protein (cf. Part A of FIG. 6 and Part B of FIG. 6)was dialyzed twice against 5 L PBS.

Homogeneous protein preparations without signs of aggregation wereobtained with a final yield of 128 mg for PA#1(200)-Crisantaspase and 48mg for PA#1(400)-Crisantaspase from one 8 L fermenter, respectively.Protein concentrations were determined by measuring the absorption at280 nm using a calculated extinction coefficient (Gill (1989) Anal.Biochem. 182: 319-326) of 19370 M⁻¹ cm⁻¹. Enzyme activities of thefusion proteins were determined using the Nessler assay described inexample 1. In this setup, the PA#1(200)-Crisantaspase fusion protein had109% and the PA#1(400)-Crisantaspase had 118% of enzyme activitycompared to the equally assayed non-modified Crisantaspase. Thisdemonstrates that the N-terminal fusion of Crisantaspase with P/Apolypeptides to the length of at least 401 amino acids does not affectenzymatic activity.

Example 6: Measurement of the Hydrodynamic Volume for Both Geneticallyand Chemically PASylated Crisantaspase by Analytical Gel Filtration

Size exclusion chromatography (SEC) was carried out on a SUPERDEX S200increase 10/300 GL column (GE Healthcare Europe, Freiburg, Germany) at aflow rate of 0.5 mL/min using an ÄKTA Purifier 10 system (GE Healthcare)with PBS (115 mM NaCl, 4 mM KH₂PO₄, 16 mM Na₂HPO₄; pH 7.4) as runningbuffer. Using regenerated cellulose disposable ultrafiltration devices(MWCO 10 kDa; Merck-Millipore, Darmstadt, Germany) recombinantCrisantaspase genetically fused with PA#1(200) or PA#1(400) polypeptides(described in Example 5) and Crisantaspase chemically conjugated witheither the Pga-P/A(40)-Ahx peptide (described in Example 2) or with thePga-P/A(20)-Ahx peptide (described in Example 3) were adjusted to aconcentration of 1 mg/mL in PBS. 150 μL samples of the concentratedPASylated enzymes and of non-PASylated Crisantaspase were individuallyapplied to the column and the chromatography traces were overlaid (PartA of FIG. 7). All five proteins eluted in single homogenous peaks.

For column calibration (Part B of FIG. 7) 150 μL of an appropriatemixture of the following globular proteins (Sigma, Deisenhofen, Germany)was applied in PBS at protein concentrations between 0.5 mg/ml and 1.0mg/ml: cytochrome c, 12.4 kDa; ovalbumin, 43.0 kDa; bovine serumalbumin, 66.3 kDa; alcohol dehydrogenase, 150 kDa; β-amylase, 200 kDa;apo-ferritin, 440 kDa; thyroglobulin, 660 kDa.

As result, both the recombinant PA fusion proteins and the chemicallyconjugated enzyme preparations exhibited a significantly larger sizethan corresponding globular proteins with the same molecular weight.With increasing size of the P/A (poly-)peptide moiety this mol.weight/hydrodynamic volume disproportion increased further. The apparentsize increase for PA(200)-Crisantaspase was 5.1-fold compared with theunfused Crisantaspase whereas the true mass was only larger by 1.5-fold.The apparent size increase for PA(400)-Crisantaspase compared with theunfused Crisantaspase was 10.4-fold whereas the true mass was onlylarger by 1.9-fold. This observation clearly indicates a much increasedhydrodynamic volume conferred to the biologically active Crisantaspaseenzyme by the Pro/Ala polypeptide segment according to this invention.

Example 7: ESI-MS Analysis of Chemically or Genetically PASylatedCrisantaspase

250 μl of the purified chemical modified protein of Crisantaspase withPga-P/A(20)-Ahx from Example 3 and of the recombinant PA200- andPA400-fusion proteins from Example 5, all at a concentration of 1 mg/mL,were applied to a 1 mL Resource™ RPC column (GE Healthcare, Freiburg,Germany) connected to an ÄKTA purifier system using 2% v/v acetonitrile,1% v/v formic acid as running buffer. The proteins were eluted using anacetonitrile gradient from 2% v/v acetonitrile, 1% v/v formic acid to80% v/v acetonitrile, 0.1% v/v formic acid over 20 column volumes. Theeluted proteins were directly analyzed via ESI mass spectrometry on aMAXIS micrOTOF instrument (Bruker Daltonik, Bremen, Germany) using thepositive ion mode. The raw m/z spectrum of theCrisantaspase/Pga-P/A(20)-Ahx chemical modified protein is shown in PartA of FIG. 8. The masses revealed by the deconvoluted mass spectrum (PartB of FIG. 8) are given in Table 3. The distribution of masses matchesthe coupling ratios determined by SD S-PAGE analysis described inExample 2.

The raw m/z spectrum of the recombinant PA#1(200)-Crisantaspase (SEQ IDNO: 11) fusion protein is shown in Part C of FIG. 8. The deconvolutedmass spectrum revealed a mass of 51164.75 Da (Part D of FIG. 8), whichessentially coincides with the calculated mass of this protein (51163.58Da). The raw m/z spectrum of the recombinant PA#1(400)-Crisantaspasefusion protein (SEQ ID NO: 13) is shown in Part E of FIG. 8. Thedeconvoluted spectrum (Part F of FIG. 8) revealed a mass of 67199.17 Da,which essentially coincides with the calculated mass of this protein(67201.99 Da). This clearly demonstrates that intact Crisantaspaseenzyme genetically fused to either PA200 or PA400 can be produced in E.coli in a highly homogeneous form.

TABLE 3 Comparison of calculated and measured masses detected in thepreparation of the Crisantaspase/Pga- P/A(20)-Ahx chemical modifiedprotein Coupling ratio Calculated mass Measured mass  9x 51506.1 51503.910x 53334.1 53333.2 11x 55162.1 55161.7 12x 56990.1 56990.1 13x 58818.158819.4 14x 60646.1 60645.8

Example 8: Asparaginase Activity

PASylated L-asparaginase enzyme activity was determined by catalysis ofthe conversion of L-asparagine into L-aspartic acid. This reactionliberates one mole of ammonia per mole of converted L-asparagine. Thereleased ammonia is detected using Nessler's reagent. In the presence ofNessler's reagent the ammonia will form a water-soluble yellow complexthat can be quantified by absorbance measurement at 450 nm (Mashburn etal. (1963) Biochem. Biophys. Res. Commun. 12, 50). One unit ofL-asparaginase enzyme activity (International Unit or IU) is defined asthe amount of enzyme that catalyzes the conversion of one μmol ofL-asparagine per minute. The specific activity of the samples (IU/mg) isdetermined by dividing the value of L-asparaginase activity expressed inIU/mL by the protein concentration expressed in mg/mL. The mass of theprotein monomer with the PASylated sequence was measured.

The measurement of L-asparaginase activity is based on an endpoint assayin which the sample is diluted to a series of final enzymeconcentrations which are then incubated at 37° C. under saturatingL-asparagine concentration for 15 minutes. The reaction is stopped byaddition of Nessler's reagent and the amount of ammonia produced by thereaction is extrapolated from a calibration curve constructed from knownquantities of ammonium sulfate used as standard. A plot of enzymeconcentration versus ammonia is then created for each sample and theslope of the curve divided by the reaction time to obtain the specificactivity in IU/mg. Specific activity is reported as IU/mg and isreported to the nearest whole number.

The initial testing results are displayed in the table below for each ofthe modified proteins or fusion proteins.

Crisantaspase Nessler Nessler Nessler Expression Plate 1 Plate 2 Plate 3Nessler System Modified protein Type (IU/mg) (IU/mg) (IU/mg) (Average)E. coli PA200-Crisantaspase 626 666 556 616 E. coliCrisantaspase-P/A(20)n 694 732 602 676 E. coli PA400-Crisantaspase 790748 699 746 E. coli Crisantaspase-P/A(40)n 567 528 490 528

Example 9: Pharmacokinetics

The pharmacokinetic profile of E. coli expressed recombinantcrisantaspase as a PASylated fusion protein (PA-200) or chemicallyconjugated to PA-peptides (PA-20) was characterized followingadministration of a single intravenous bolus dose to CD-1 mice. The CD-1mice is a model for a healthy mouse.

All animals received a single intravenous (IV) bolus via the lateraltail vein (10 mL/kg) based on the body weight taken prior to dosing.Individual doses were calculated based upon the most recent individualbody weights to provide the proper dose. The first day of dosing wasbased on study day 0 body weights. All animals were observed formortality, abnormalities, and signs of pain and distress twice daily,once in the morning and once in the afternoon.

PASylated asparaginase was administered as a single IV dose of 25 IU/kgbody weight to mice. Groups of mice were dosed at 25 IU/kg body weightand plasma samples were collected at scheduled times for up to 10 days(240 h) following dosing. Asparaginase activity in mouse plasma wasmeasured using a qualified biochemical assay as described in theprevious examples. Mean plasma asparaginase activity (n=4) versus timedata are plotted (FIG. 1) and pharmacokinetic analyses were conducted.

Blood samples were taken prior to dosing and at approximately 6, 24 (Day1), 48 (Day 2), 51 (Day 2), 54 (Day 2), 60 (Day 2), 96 (Day 4), 168 (Day7), and 240 (Day 10) hours post dose. Tail-snip (cut end of tail) bloodcollection procedure was employed. Approximately 1 to 2 mm was cut offthe distal end of the tail for the first blood collection, allsequential blood collections were collected from the same site byremoving the scab and facilitating blood flow by stroking the tail.Approximately 100 μL blood per time point was collected into chilledK₃EDTA (Minivette) sampling tubes. Blood was transferred into tubesappropriate for centrifugation. For plasma isolation, all samples werecentrifuged within approximately 20 minutes of sampling at 3,000×g in arefrigerated centrifuge set to maintain approximately 4° C. forapproximately 10 minutes. Following centrifugation, the maximum amountof plasma was recovered (targeting 30 μL) and placed into plastic vials.The plastic vials were stored at −65° C. to −85° C. until testing.

Asparaginase activity was measured as the concentration of asparaginasein the plasma samples as previously described (Alias et al. (2009)Blood, 114, 2033). Parameters dependent on sufficient characterizationof the terminal phase of the concentration versus time profile (t½, CL,and V_(ss)) were only reported if R² (the square of the correlationcoefficient for linear regression used to estimate the terminalelimination rate constant, λz) was greater than 0.8. The pharmacokineticdata was imported into Phoenix WinNonlin v6.4 (Certara/Pharsight) foranalysis. The plasma asparaginase activity versus time data wereanalyzed using non-compartmental methods with sparse sampling in an IVbolus administration model. Activity values below the limit ofquantitation of the assay (10 U/L) were set to zero in the calculationof group means. Nominal dose levels and sample collection times wereused for the calculations. The estimated t1/2 values were 50.2 h forPA-20 crisantaspase and 17.9 h for PA-200 crisantaspase.

The present invention refers to the following nucleotide and amino acidsequences:

Some sequences provided herein are available in the NCBI database andcan be retrieved from ncbi.nlm.nih.gov/sites/entrez?db=gene; Thesessequences also relate to annotated and modified sequences. The presentinvention also provides techniques and methods wherein homologoussequences, and variants of the concise sequences provided herein areused. Preferably, such “variants” are genetic variants.

SEQ ID NO: 1:Amino acid sequence of Dickeya chrysanthemi L-Asparaginase.ADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGEQFSNMASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTVKSDKPVVFVAAMRPATAISADGPMNLLEAVRVAGDKQSRGRGVMVVLNDRIGSARYITKTNASTLDTFKANEEGYLGVIIGNRIYYQNRIDKLHTTRSVFDVRGLTSLPKVDILYGYQDDPEYLYDAAIQHGVKGIVYAGMGAGSVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEELPGLVSDSLNPAHARILLMLALTRTSDPKVIQEYFHTY SEQ ID NO: 2:Nucleotide sequence encoding Dickeya chrysanthemi L-AsparaginaseGCAGATAAACTGCCGAATATTGTTATTCTGGCAACCGGTGGCACCATTGCAGGTAGCGCAGCAACCGGCACCCAAACCACAGGTTATAAAGCCGGTGCACTGGGTGTTGATACCCTGATTAATGCAGTTCCGGAAGTTAAAAAACTGGCCAATGTGAAAGGTGAACAGTTTAGCAATATGGCCAGCGAAAATATGACCGGTGATGTTGTTCTGAAACTGAGCCAGCGTGTTAATGAACTGCTGGCACGTGATGATGTTGATGGTGTGGTTATTACCCATGGCACCGATACCGTTGAAGAAAGCGCCTATTTTCTGCATCTGACCGTGAAAAGCGATAAACCGGTTGTTTTTGTTGCAGCAATGCGTCCGGCAACCGCAATTAGCGCAGATGGTCCGATGAATCTGCTGGAAGCAGTTCGTGTTGCCGGTGATAAACAGAGCCGTGGTCGTGGTGTTATGGTTGTTCTGAATGATCGTATTGGTAGCGCACGCTATATTACCAAAACCAATGCAAGCACCCTGGATACCTTTAAAGCCAATGAAGAAGGTTATCTGGGCGTTATTATTGGCAATCGCATTTATTATCAGAATCGCATTGATAAACTGCATACCACCCGTAGCGTTTTTGATGTTCGTGGTCTGACCAGCCTGCCGAAAGTTGATATTCTGTATGGCTATCAGGATGATCCGGAATATCTGTATGATGCAGCCATTCAGCATGGTGTTAAAGGTATTGTGTATGCAGGTATGGGTGCAGGTAGCGTTAGCGTTCGTGGTATTGCAGGTATGCGTAAAGCAATGGAAAAAGGCGTTGTTGTTATTCGTAGCACCCGTACCGGTAATGGTATTGTTCCGCCGGATGAAGAACTGCCGGGTCTGGTTAGCGATAGCCTGAATCCGGCACATGCACGTATTCTGCTGATGCTGGCACTGACCCGTACCAGCGATCCGAAAGTGATTCAGGAATATTTTCATACCTAT SEQ ID NO: 3:Amino acid sequence of Dickeya chrysanthemi L-AsparaginaseSignal peptide: 1-28; removed during cloning: 29-39; 40-366 asparaginaseMFKFKKNFLVGLSAALMSISLFSATASA ARRAIVGRSSAADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGEQFSNMASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTVKSDKPVVFVAAMRPATAISADGPMNLLEAVRVAGDKQSRGRGVMVVLNDRIGSARYITKTNASTLDTFKANEEGYLGVIIGNRIYYQNRIDKLHTTRSVFDVRGLTSLPKVDILYGYQDDPEYLYDAAIQHGVKGIVYAGMGAGSVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEELPGLVSDSLNPAHARILLMLALTRTSDPKVIQEYFHTY SEQ ID NO: 4Nucleotide sequence (synthetic) encoding Dickeya chrysanthemi L-Asparaginasemature asparaginase coded from base 160-1140 (bold letters). Thus, a nucleotidesequence encoding L-Asparaginase ranges from nucleotides at position 160 to 1140.TCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGTTCAAATTCAAAAAAAACTTCCTGGTGGGTCTGAGCGCAGCACTGATGAGCATTAGCCTGTTTAGCGCAACCGCAAGCGCAGCCAGAAGAGCGATTGTAGGACGCTCTTCTGCCGCAGATAAACTGCCGAATATTGTTATTCTGGCAACCGGTGGCACCATTGCAGGTAGCGCAGCAACCGGCACCCAGACCACCGGTTATAAAGCCGGTGCACTGGGTGTTGATACCCTGATTAATGCAGTTCCGGAAGTTAAAAAACTGGCCAATGTTAAAGGTGAGCAGTTTAGCAATATGGCCAGCGAAAATATGACCGGTGATGTTGTTCTGAAACTGAGCCAGCGTGTTAATGAACTGCTGGCACGTGATGATGTTGATGGTGTTGTTATTACCCATGGCACCGATACCGTTGAAGAAAGCGCATATTTTCTGCATCTGACCGTGAAAAGCGATAAACCGGTTGTTTTTGTTGCAGCAATGCGTCCGGCAACCGCCATTAGCGCAGATGGTCCGATGAATCTGCTGGAAGCAGTTCGTGTTGCCGGTGATAAACAGAGCCGTGGTCGTGGTGTTATGGTTGTGCTGAATGATCGTATTGGTAGCGCACGTTATATTACCAAAACCAATGCAAGCACCCTGGATACCTTTAAAGCAAATGAAGAAGGTTATCTGGGCGTCATTATTGGCAATCGTATCTATTATCAGAACCGCATCGACAAACTGCATACCACCCGTAGCGTTTTTGATGTTCGTGGTCTGACCAGCCTGCCGAAAGTGGATATTCTGTATGGTTATCAGGATGATCCGGAATATCTGTATGATGCAGCAATTCAGCATGGTGTGAAAGGTATTGTTTATGCAGGTATGGGTGCGGGTAGCGTTAGCGTTCGTGGTATTGCCGGTATGCGTAAAGCAATGGAAAAAGGTGTTGTTGTGATTCGTAGCACCCGTACCGGTAATGGTATTGTTCCGCCTGATGAAGAACTGCCTGGTCTGGTTAGCGATAGCCTGAATCCGGCACATGCACGTATTCTGCTGATGCTGGCACTGACCCGTACCAGCGATCCGAAAGTTATTCAAGAATATTTTCATACCTATTA AGCTTSEQ ID NO: 5: Amino acid sequence of PA(20) peptide AAPAAPAPAAPAAPAPAAPASEQ ID NO: 6: Nucleotide sequence encoding PA(20) peptideGCCGCGCCAGCGGCCCCGGCCCCTGCCGCGCCCGCTGCTCCCGCCCCTGCTGCCCCAGCCSEQ ID NO: 7: Amino acid sequence of PA(200)-polypeptideAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAA SEQ ID NO: 8:Nucleotide sequence encoding PA(200)-polypeptideGCCGCGCCAGCGGCCCCGGCCCCTGCCGCGCCCGCTGCTCCCGCCCCTGCTGCCCCAGCCGCCGCTCCTGCGGCACCTGCGCCCGCCGCGCCGGCAGCGCCGGCACCGGCAGCTCCGGCGGCCGCGCCTGCAGCTCCTGCACCGGCGGCTCCAGCAGCCCCGGCGCCGGCCGCACCTGCGGCGGCGCCCGCGGCGCCTGCACCCGCAGCGCCTGCGGCACCGGCCCCAGCAGCCCCTGCCGCCGCACCGGCTGCGCCTGCCCCAGCGGCCCCCGCTGCCCCGGCCCCGGCGGCTCCAGCCGCAGCGCCTGCCGCCCCAGCGCCCGCAGCACCGGCGGCACCAGCTCCGGCGGCGCCGGCGGCGGCTCCGGCAGCTCCGGCCCCTGCTGCGCCGGCTGCGCCGGCTCCGGCGGCCCCTGCGGCGGCTCCGGCCGCACCTGCACCTGCCGCGCCGGCTGCTCCGGCCCCGGCTGCCCCAGCAGCGGCACCAGCAGCGCCTGCTCCTGCGGCGCCTGCAGCTCCGGCGCCGGCAGCCCCGGCCGCCGCACCCGCGGCTCCAGCCCCCGCCGCTCCAGCAGCCCCCGCGCCAGCTGCACC TGCTGCCSEQ ID NO: 9: Amino acid sequence of PA(400)-polypeptideAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAA SEQ ID NO: 10:Nucleotide sequence encoding PA(400)-polypeptideGCCGCGCCAGCGGCCCCGGCCCCTGCCGCGCCCGCTGCTCCCGCCCCTGCTGCCCCAGCCGCCGCTCCTGCGGCACCTGCGCCCGCCGCGCCGGCAGCGCCGGCACCGGCAGCTCCGGCGGCCGCGCCTGCAGCTCCTGCACCGGCGGCTCCAGCAGCCCCGGCGCCGGCCGCACCTGCGGCGGCGCCCGCGGCGCCTGCACCCGCAGCGCCTGCGGCACCGGCCCCAGCAGCCCCTGCCGCCGCACCGGCTGCGCCTGCCCCAGCGGCCCCCGCTGCCCCGGCCCCGGCGGCTCCAGCCGCAGCGCCTGCCGCCCCAGCGCCCGCAGCACCGGCGGCACCAGCTCCGGCGGCGCCGGCGGCGGCTCCGGCAGCTCCGGCCCCTGCTGCGCCGGCTGCGCCGGCTCCGGCGGCCCCTGCGGCGGCTCCGGCCGCACCTGCACCTGCCGCGCCGGCTGCTCCGGCCCCGGCTGCCCCAGCAGCGGCACCAGCAGCGCCTGCTCCTGCGGCGCCTGCAGCTCCGGCGCCGGCAGCCCCGGCCGCCGCACCCGCGGCTCCAGCCCCCGCCGCTCCAGCAGCCCCCGCGCCAGCTGCACCTGCTGCCGCTCCTGCTGCCCCTGCTCCCGCTGCCCCCGCCGCCCCCGCCCCAGCTGCCCCCGCTGCCGCACCTGCTGCCCCAGCTCCCGCTGCCCCAGCCGCGCCGGCCCCCGCAGCTCCAGCCGCGGCACCAGCTGCCCCAGCTCCAGCGGCGCCTGCTGCCCCGGCCCCCGCGGCACCGGCTGCCGCGCCCGCAGCTCCAGCGCCTGCTGCACCGGCTGCTCCGGCACCCGCCGCGCCAGCAGCTGCCCCTGCGGCACCAGCTCCTGCTGCCCCCGCGGCACCTGCACCCGCTGCCCCGGCGGCAGCTCCCGCCGCGCCAGCCCCTGCAGCTCCTGCTGCACCTGCTCCTGCCGCCCCTGCTGCTGCCCCTGCTGCTCCAGCCCCTGCAGCACCGGCCGCTCCAGCTCCTGCCGCTCCTGCCGCTGCGCCCGCTGCTCCAGCCCCAGCTGCGCCAGCAGCTCCTGCACCTGCTGCCCCTGCCGCCGCCCCTGCGGCTCCAGCACCTGCTGCACCGGCCGCCCCGGCGCCCGCTGCCCCCGCAGCAGCCCCAGCCGCACCCGCTCCAGCAGCTCCCGCAGCCCCAGCACCCGCAGCACCA GCCGCCSEQ ID NO: 11:Amino acid sequence of Asparaginase-PA(200)-fusion proteinAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGEQFSNMASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTVKSDKPVVFVAAMRPATAISADGPMNLLEAVRVAGDKQSRGRGVMVVLNDRIGSARYITKTNASTLDTFKANEEGYLGVIIGNRIYYQNRIDKLHTTRSVFDVRGLTSLPKVDILYGYQDDPEYLYDAAIQHGVKGIVYAGMGAGSVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEELPGLVSDSLNPAHARILLMLALTRTSDPKVIQEYFHTY SEQ ID NO: 12:Nucleotide sequence encoding Asparaginase-PA(200)-fusion protein (XbaI/HindIII)Mature fusion protein (SEQ ID NO: 11) coded from base 127-1710 (bold letters).Thus, a nucleotide sequence encoding a fusion protein can range from nucleotidesat position 127 to 1710 of SEQ ID NO: 12. Accordingly, the term “modified proteincomprising or consisting of an amino acid sequence encoded by a nucleic acidhaving a nucleotide sequence as shown in SEQ ID NO: 12” as used hereincan be more narrowly defined as “modified protein comprising or consisting ofan amino acid sequence encoded by a nucleic acid having a nucleotide sequence asshown in positions 127 to 1710 of SEQ ID NO: 12”.TCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGTTCAAATTCAAAAAAAACTTCCTGGTGGGTCTGAGCGCAGCACTGATGAGCATTAGCCTGTTTAGCGCAACCGCAAGCGCAGCCGCGCCAGCGGCCCCGGCCCCTGCCGCGCCCGCTGCTCCCGCCCCTGCTGCCCCAGCCGCCGCTCCTGCGGCACCTGCGCCCGCCGCGCCGGCAGCGCCGGCACCGGCAGCTCCGGCGGCCGCGCCTGCAGCTCCTGCACCGGCGGCTCCAGCAGCCCCGGCGCCGGCCGCACCTGCGGCGGCGCCCGCGGCGCCTGCACCCGCAGCGCCTGCGGCACCGGCCCCAGCAGCCCCTGCCGCCGCACCGGCTGCGCCTGCCCCAGCGGCCCCCGCTGCCCCGGCCCCGGCGGCTCCAGCCGCAGCGCCTGCCGCCCCAGCGCCCGCAGCACCGGCGGCACCAGCTCCGGCGGCGCCGGCGGCGGCTCCGGCAGCTCCGGCCCCTGCTGCGCCGGCTGCGCCGGCTCCGGCGGCCCCTGCGGCGGCTCCGGCCGCACCTGCACCTGCCGCGCCGGCTGCTCCGGCCCCGGCTGCCCCAGCAGCGGCACCAGCAGCGCCTGCTCCTGCGGCGCCTGCAGCTCCGGCGCCGGCAGCCCCGGCCGCCGCACCCGCGGCTCCAGCCCCCGCCGCTCCAGCAGCCCCCGCGCCAGCTGCACCTGCTGCCGCAGATAAACTGCCGAATATTGTTATTCTGGCAACCGGTGGCACCATTGCAGGTAGCGCAGCAACCGGCACCCAGACCACCGGTTATAAAGCCGGTGCACTGGGTGTTGATACCCTGATTAATGCAGTTCCGGAAGTTAAAAAACTGGCCAATGTTAAAGGTGAGCAGTTTAGCAATATGGCCAGCGAAAATATGACCGGTGATGTTGTTCTGAAACTGAGCCAGCGTGTTAATGAACTGCTGGCACGTGATGATGTTGATGGTGTTGTTATTACCCATGGCACCGATACCGTTGAAGAAAGCGCATATTTTCTGCATCTGACCGTGAAAAGCGATAAACCGGTTGTTTTTGTTGCAGCAATGCGTCCGGCAACCGCCATTAGCGCAGATGGTCCGATGAATCTGCTGGAAGCAGTTCGTGTTGCCGGTGATAAACAGAGCCGTGGTCGTGGTGTTATGGTTGTGCTGAATGATCGTATTGGTAGCGCACGTTATATTACCAAAACCAATGCAAGCACCCTGGATACCTTTAAAGCAAATGAAGAAGGTTATCTGGGCGTCATTATTGGCAATCGTATCTATTATCAGAACCGCATCGACAAACTGCATACCACCCGTAGCGTTTTTGATGTTCGTGGTCTGACCAGCCTGCCGAAAGTGGATATTCTGTATGGTTATCAGGATGATCCGGAATATCTGTATGATGCAGCAATTCAGCATGGTGTGAAAGGTATTGTTTATGCAGGTATGGGTGCGGGTAGCGTTAGCGTTCGTGGTATTGCCGGTATGCGTAAAGCAATGGAAAAAGGTGTTGTTGTGATTCGTAGCACCCGTACCGGTAATGGTATTGTTCCGCCTGATGAAGAACTGCCTGGTCTGGTTAGCGATAGCCTGAATCCGGCACATGCACGTATTCTGCTGATGCTGGCACTGACCCGTACCAGCGATCCGAAAGTTATTCAAGAATATTTTCATACCTATTAAGCTT SEQ ID NO: 13:Amino acid sequence of Asparaginase-PA(400)-fusion proteinAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGEQFSNMASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTVKSDKPVVFVAAMRPATAISADGPMNLLEAVRVAGDKQSRGRGVMVVLNDRIGSARYITKTNASTLDTFKANEEGYLGVIIGNRIYYQNRIDKLHTTRSVFDVRGLTSLPKVDILYGYQDDPEYLYDAAIQHGVKGIVYAGMGAGSVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEELPGLVSDSLNPAHARILLMLALTRTSDPKVIQEYFHTY SEQ ID NO: 14:Nucleotide sequence encoding Asparaginase-PA(400)-fusion protein(XbaI/HindIII)Mature fusion protein (SEQ ID NO: 13) coded from base 127-2184 (bold letters).Thus, a nucleotide sequence encoding a fusion protein can range from nucleotidesat position 127 to 2184 of SEQ ID NO: 14. Accordingly, the term “modified proteincomprising or consisting of an amino acid sequence encoded by a nucleic acid havinga nucleotide sequence as shown in SEQ ID NO: 14”as used herein can be more narrowlydefined as “modified protein comprising or consisting of an amino acid sequenceencoded by a nucleic acid having a nucleotide sequence as shown in positions 127 to2184 of SEQ ID NO: 14”.TCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGTTCAAATTCAAAAAAAACTTCCTGGTGGGTCTGAGCGCAGCACTGATGAGCATTAGCCTGTTTAGCGCAACCGCAAGCGCAGCCGCGCCAGCGGCCCCGGCCCCTGCCGCGCCCGCTGCTCCCGCCCCTGCTGCCCCAGCCGCCGCTCCTGCGGCACCTGCGCCCGCCGCGCCGGCAGCGCCGGCACCGGCAGCTCCGGCGGCCGCGCCTGCAGCTCCTGCACCGGCGGCTCCAGCAGCCCCGGCGCCGGCCGCACCTGCGGCGGCGCCCGCGGCGCCTGCACCCGCAGCGCCTGCGGCACCGGCCCCAGCAGCCCCTGCCGCCGCACCGGCTGCGCCTGCCCCAGCGGCCCCCGCTGCCCCGGCCCCGGCGGCTCCAGCCGCAGCGCCTGCCGCCCCAGCGCCCGCAGCACCGGCGGCACCAGCTCCGGCGGCGCCGGCGGCGGCTCCGGCAGCTCCGGCCCCTGCTGCGCCGGCTGCGCCGGCTCCGGCGGCCCCTGCGGCGGCTCCGGCCGCACCTGCACCTGCCGCGCCGGCTGCTCCGGCCCCGGCTGCCCCAGCAGCGGCACCAGCAGCGCCTGCTCCTGCGGCGCCTGCAGCTCCGGCGCCGGCAGCCCCGGCCGCCGCACCCGCGGCTCCAGCCCCCGCCGCTCCAGCAGCCCCCGCGCCAGCTGCACCTGCTGCCGCTCCTGCTGCCCCTGCTCCCGCTGCCCCCGCCGCCCCCGCCCCAGCTGCCCCCGCTGCCGCACCTGCTGCCCCAGCTCCCGCTGCCCCAGCCGCGCCGGCCCCCGCAGCTCCAGCCGCGGCACCAGCTGCCCCAGCTCCAGCGGCGCCTGCTGCCCCGGCCCCCGCGGCACCGGCTGCCGCGCCCGCAGCTCCAGCGCCTGCTGCACCGGCTGCTCCGGCACCCGCCGCGCCAGCAGCTGCCCCTGCGGCACCAGCTCCTGCTGCCCCCGCGGCACCTGCACCCGCTGCCCCGGCGGCAGCTCCCGCCGCGCCAGCCCCTGCAGCTCCTGCTGCACCTGCTCCTGCCGCCCCTGCTGCTGCCCCTGCTGCTCCAGCCCCTGCAGCACCGGCCGCTCCAGCTCCTGCCGCTCCTGCCGCTGCGCCCGCTGCTCCAGCCCCAGCTGCGCCAGCAGCTCCTGCACCTGCTGCCCCTGCCGCCGCCCCTGCGGCTCCAGCACCTGCTGCACCGGCCGCCCCGGCGCCCGCTGCCCCCGCAGCAGCCCCAGCCGCACCCGCTCCAGCAGCTCCCGCAGCCCCAGCACCCGCAGCACCAGCCGCCGCAGATAAACTGCCGAATATTGTTATTCTGGCAACCGGTGGCACCATTGCAGGTAGCGCAGCAACCGGCACCCAGACCACCGGTTATAAAGCCGGTGCACTGGGTGTTGATACCCTGATTAATGCAGTTCCGGAAGTTAAAAAACTGGCCAATGTTAAAGGTGAGCAGTTTAGCAATATGGCCAGCGAAAATATGACCGGTGATGTTGTTCTGAAACTGAGCCAGCGTGTTAATGAACTGCTGGCACGTGATGATGTTGATGGTGTTGTTATTACCCATGGCACCGATACCGTTGAAGAAAGCGCATATTTTCTGCATCTGACCGTGAAAAGCGATAAACCGGTTGTTTTTGTTGCAGCAATGCGTCCGGCAACCGCCATTAGCGCAGATGGTCCGATGAATCTGCTGGAAGCAGTTCGTGTTGCCGGTGATAAACAGAGCCGTGGTCGTGGTGTTATGGTTGTGCTGAATGATCGTATTGGTAGCGCACGTTATATTACCAAAACCAATGCAAGCACCCTGGATACCTTTAAAGCAAATGAAGAAGGTTATCTGGGCGTCATTATTGGCAATCGTATCTATTATCAGAACCGCATCGACAAACTGCATACCACCCGTAGCGTTTTTGATGTTCGTGGTCTGACCAGCCTGCCGAAAGTGGATATTCTGTATGGTTATCAGGATGATCCGGAATATCTGTATGATGCAGCAATTCAGCATGGTGTGAAAGGTATTGTTTATGCAGGTATGGGTGCGGGTAGCGTTAGCGTTCGTGGTATTGCCGGTATGCGTAAAGCAATGGAAAAAGGTGTTGTTGTGATTCGTAGCACCCGTACCGGTAATGGTATTGTTCCGCCTGATGAAGAACTGCCTGGTCTGGTTAGCGATAGCCTGAATCCGGCACATGCACGTATTCTGCTGATGCTGGCACTGACCCGTACCAGCGATCCGAAAGTTATTCAAGAATATTTTCATACCTATT AAGCTTSEQ ID NO: 15. Amino acid sequence of PA(40) peptideAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPASEQ ID NO: 16: Modified PA(20) peptide Pga-AAPAAPAPAAPAAPAPAAPA-Ahx-COOHSEQ ID NO: 17: Modified PA(40) peptidePga-AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA-Ahx-COOH

All references cited herein are fully incorporated by reference. Havingnow fully described the invention, it will be understood by a personskilled in the art that the invention may be practiced within a wide andequivalent range of conditions, parameters and the like, withoutaffecting the spirit or scope of the invention or any embodimentthereof.

1. A modified fusion protein comprising (i) an L-asparaginase comprisingthe amino acid sequence of SEQ ID NO: 1, and (ii) one or morepolypeptides, wherein the one or more polypeptides comprise the aminoacid sequence of SEQ ID NO: 7 or
 9. 2-5. (canceled)
 6. The modifiedfusion protein according to claim 1, wherein the L-asparaginase consistsof the amino acid sequence of SEQ ID NO:
 1. 7. The modified fusionprotein according to claim 1, wherein the modified protein is a fusionprotein of the L-asparaginase and the one or more polypeptides.
 8. Themodified fusion protein according to claim 7, wherein the one or morepolypeptides consist of 200 to 600 proline and alanine amino acidresidues.
 9. The modified fusion protein according to claim 7, whereinthe one or more polypeptides consist of 200 to 400 proline and alanineamino acid residues, and the proline amino acid residues constitute morethan 10% and less than 70% of the polypeptide.
 10. (canceled)
 11. Themodified fusion protein according to claim 7, wherein at least one ofthe one or more polypeptides comprises the amino acid sequence of SEQ IDNO:
 7. 12. The modified fusion protein according to claim 7, wherein atleast one of the one or more polypeptides comprises the amino acidsequence of SEQ ID NO:
 9. 13. The modified fusion protein according toclaim 7, wherein the fusion protein comprises the amino acid sequence ofSEQ ID NO:
 11. 14. The modified fusion protein according to claim 7,wherein the fusion protein comprises the amino acid sequence of SEQ IDNO:
 13. 15. The modified fusion protein according to claim 7, whereinthe L-asparaginase comprises the amino acid sequence of SEQ ID NO: 1 andthe polypeptide consists of the amino acid sequence of SEQ ID NO: 7, or9. 16-17. (canceled)
 19. The modified fusion protein according to claim6, wherein at least one of the one or more polypeptides consists of theamino acid sequence of SEQ ID NO:
 7. 20. The modified fusion proteinaccording to claim 6, wherein at least one of the one or morepolypeptides consists of the amino acid sequence of SEQ ID NO:
 9. 21-22.(canceled)
 23. The modified fusion protein according to claim 7, whereinthe L-asparaginase is composed of four subunits.
 24. The modified fusionprotein according to claim 7, wherein the fusion protein consists of theamino acid sequence of SEQ ID NO: 11 or
 13. 25. A pharmaceuticalcomposition comprising the modified fusion protein according to claim 1and one or more pharmaceutical acceptable carriers or excipients. 26.The pharmaceutical composition of claim 25, wherein the one or morepolypeptides is present in an amount sufficient to mediate a decreasedimmunogenicity of the modified protein following administration to ahuman subject.
 27. The pharmaceutical composition of claim 25, whereinthe one or more polypeptides is present in an amount sufficient toincrease the plasma half-life of the L-asparaginase followingadministration to a human subject.
 28. (canceled)